Immunogenic proteins of Leptospira

ABSTRACT

The invention provides novel immunogenic proteins LigA and LigB from  Leptospira  for use in the development of effective vaccines and antibodies, as well as improved diagnostic methods and kits.

RELATED APPLICATION

This application is a continuation under 35 U.S.C. 111(a) ofInternational Application No. PCT/US03/32385 filed Oct. 10, 2003 andpublished in English as WO 2004/032599 A3 on Apr. 22, 2004, which claimspriority under 35 USC 119(e) from U.S. Provisional Application Ser. No.60/417,721, filed Oct. 10, 2002, which applications and publication areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Leptospirosis is a worldwide zoonotic disease caused by gram-negativespirochetes belonging to the genus Leptospira. Leptospirosis isprevalent in humans, horses, cattle and wild animals. The disease occurswidely in developing countries, such as Brazil and India, and isre-emerging in developed countries. Although the incidence ofleptospirosis in humans in the United States is relatively low, diseaseincidence in domestic animals has increased in recent years.

Manifestations and routes of Leptospira infection vary depending on thehost. Humans, who contract leptospirosis either directly or indirectlythrough contact with infected animals or a contaminated environment,often develop kidney and liver failure (Schubert, G. E. et al., MunchMed Wochenschr, 113:80-86 (1971); Bain, B. J. et al., Arch. Intern.Med., 131:740-745 (1973); Garcia, M. et al., Med. Clin. (Barc),73:362-366 (1979); San Segundo, D., Med. Clin. (Barc), 78:28-31 (1982);Winearls, C. G. et al., Q J Med., 53:487-495 (1984); Menzies, D. G. etal., Scott Med. J., 34:410 (1989); Divers, T. J. et al., J. Am. Vet.Med. Assoc., 201:1391-1392 (1992); Petros, S. et al., Scand. J. Infect.Dis., 32:104-105 (2000); Kager, P. A. et al., Ned Tijdschr Geneeskd,145:184-186 (2001)). Leptospira infection in humans can range inseverity from an inapparent infection to death from renal or hepaticfailure (Feigin, R. D. and D. C. Anderson, CRC Crit. Rev. Clin. Lab.Sci., 5:413-467 (1975)). In addition to hepatic and renal failure,uveitis is sometimes a sequela to Leptospira infection (Rathinam, S. R.et al., Am. J. Ophthalmol., 124:71-79 (1997)).

In animals such as horses, cattle, dogs and swine, infection causesabortion, still birth, renal failure, and uveitis (Akkermans, J. P.,Bull. Off. Int. Epizoot., 66:849-866 (1966); Ellis, W. A. et al., Vet.Rec., 99:458-459 (1976); Ryan, T. J. et al., NZ Vet. J., 25:352 (1977);Ellis, W. A. et al., Vet. Rec., 103:237-239 (1978); Andreani, E. et al.,Br. Vet. J., 139:165-170 (1983); Ellis, W. A. et al., Vet. Rec.,112:291-293 (1983); Elder, J. K. et al., Aust. Vet. J., 62:258-262(1985); Ellis, W. A. et al., Vet. Rec., 118:294-295 (1986); Rocha, T.,Vet. Rec., 126:602 (1990); Bolin, C. A. et al., J. Vet. Diagn. Invest.,3:152-154 (1991); Donahue, J. M. et al., J. Vet. Diagn. Invest.,3:148-151 (1991); Christianson, W. T., Vet. Clin. North Am. Food Anim.Pract., 8:623-639 (1992); Donahue, J. M. et al., J. Vet. Diagn. Invest.,4:279-284 (1992); Bernard, W. V. et al., J. Am. Vet. Med. Assoc.,202:1285-1286 (1993); Broll, S. et al., Zentralbl Veterinarmed [B],40:641-653 (1993); Donahue, J. M. et al., J. Vet. Diagn. Invest.,7:87-91 (1995); Donahue, J. M. and Williams, N. M., Vet. Clin. North Am.Equine Pract., 16:443-456 (2000)) and can result in multi-organ failure.In horses, the most common manifestations of infection are abortion anduveitis (Poonacha, K. B. et al., Vet. Pathol., 30:362-369 (1993)). Theassociation of leptospires with equine recurrent uveitis (ERU)(Halliwell, R. E. et al., Curr. Eye Res., 4:1033-1040 (1985)) has beenwell documented and the organism has been detected in ocular fluids byculture and PCR (Roberts, S. J., J. Amer. Vet. Med. Assoc., 175:803-808(1958)). In addition, Parma et al. demonstrated reactivity of severalbands in extracts of equine cornea and lens with anti-leptospiral seraby western blotting (Parma, A. E. et al., Vet. Immunol. Immunopathol.,14:181-185 (1987); Parma, A. E. et al., Vet. Immunol. Immunopathol.,10:215-224 (1985)). Although there is a strong association betweenleptospiral infection and uveitis, the immunopathogenesis ofLeptospira-associated uveitis remains obscure.

Currently available leptospiral vaccines have low efficacy, are serovarspecific and generally produce only short-term immunity in domesticlivestock. In fact, these vaccines do not provide cross protectionagainst the 250 known serovars of pathogenic Leptospira. Efforts toidentify immunogenic components of value in vaccines have resulted incharacterization of 31, 32, 36 and 41 kDa outer membrane proteins(Haake, D. A. et al., J. Bacteriol., 175:4225-4234 (1993); Haake, D. A.et al., Infect. Immun., 68:2276-2285 (2000); Haake, D. A. et al.,Infect. Immun., 66:1579-1587 (1998); Haake, D. A. et al., Infect.Immun., 67:6572-6582 (1999); Shang, E. S. et al., Infect. Immun.,65:3174-3181 (1995); Shang, E. S. et al., Infect. Immun., 64:2322-2330(1996)). Two of these proteins (31 and 41 kDa) function synergisticallyin immunoprotection of hamsters suggesting that an effective proteinbased vaccine would contain several components (Haake, D. A. et al.,Infect. Immun., 68:2276-2285 (2000)). The search for protectiveimmunogens is complicated by the possibility that important componentsmay be produced only during infection. Supporting this possibility arerecent studies that indicate that some immunogenic proteins of L.interrogans serovar pomona are upregulated at 37° C. (Nally, J. E. etal., Infect. Immun., 69:400-404 (2001)).

Thus, there is an ongoing need for novel immunogenic proteins ofLeptospira to aid in the development of effective vaccines andantibodies, as well as improved diagnostic methods and kits.

SUMMARY OF THE INVENTION

The present invention provides an isolated and purified polynucleotidecomprising a nucleic acid sequence encoding ligA from Leptospirainterrogans. Also provided by the present invention is thepolynucleotide comprising SEQ ID NO: 1.

The invention further provides an isolated and purified polynucleotidecomprising a nucleic acid sequence encoding ligB from Leptospirainterrogans. Also provided by the present invention is thepolynucleotide comprising SEQ ID NO: 3 and SEQ ID NO: 45.

An isolated and purified polypeptide comprising a LigA polypeptide fromLeptospira interrogans is provided by the present invention. Thepolypeptide comprising SEQ ID NO: 2 is also provided by the presentinvention

The present invention provides an isolated and purified polypeptidecomprising a LigB polypeptide from Leptospira interrogans. Further, theinvention provides the polypeptide comprising SEQ ID NO: 4 and SEQ IDNO: 46.

The present invention provides a pharmaceutical composition comprising apurified polypeptide from Leptospira, and a pharmaceutically acceptablecarrier, wherein the composition is capable of eliciting an immuneresponse against Leptospira interrogans. The polypeptide in thepharmaceutical composition of the present invention may comprise thepolypeptides LigA or LigB. The pharmaceutical composition of the presentinvention may also optionally comprise an effective amount of animmunological adjuvant.

The present invention provides a vaccine comprising an immunogenicamount of a purified polypeptide from Leptospira, wherein thepolypeptide is present in an amount that is effective to immunize asusceptible mammal against Leptospira infection in combination with aphysiologically acceptable, non-toxic vehicle.

The polypeptide in the vaccine of the present invention may be LigA orLigB. The vaccine may also comprise an effective amount of animmunological adjuvant, and may be administered orally, mucosally, or bysubcutaneous or intramuscular injection.

Further provided by the present invention is a method of eliciting animmune response in a subject against Leptospira interrogans, comprisingadministering to a subject the pharmaceutical composition describedhereinabove. Another method provided by the present invention is amethod of preventing Leptospira interrogans infections comprisingadministering to a subject the pharmaceutical composition describedhereinabove.

The present invention additionally provides a method of protecting asusceptible mammal against Leptospira infection or colonizationcomprising administering to the mammal an effective amount of a vaccinecomprising an immunogenic amount of Leptospira protein LigA or LigBwherein the amount of LigA or LigB is effective to immunize thesusceptible mammal against Leptospira in combination with aphysiologically-acceptable, non-toxic vehicle.

The present invention provides a composition comprising an amount of animmunologically active protein comprising SEQ ID NO:2, SEQ ID NO: 4, atleast 9 amino acids of SEQ ID NO: 2, or at least 9 amino acids of SEQ IDNO: 4, and a pharmaceutically acceptable carrier, which amount iseffective to stimulate the formation of antibodies against Leptospirainterrogans in a mammal, e.g., a human. Further provided by the presentinvention is a composition comprising an amount of an immunologicallyactive protein comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 46, atleast 9 amino acids of SEQ ID NO: 2, at least 9 amino acids of SEQ IDNO: 4, or at least 9 amino acids of SEQ ID NO: 46, which amount iseffective to immunize a susceptible mammal against infection caused byLeptospira. The invention also provides that a composition as describedin this paragraph is effective as a vaccine. The invention additionallyprovides that a composition as described in this paragraph furthercomprises an effective amount of an immune stimulating agent.

The present invention provides a method of stimulating the formation ofantibodies against Leptospira, comprising administering to a mammal acomposition comprising an effective amount of an immunologically activeprotein having SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 46, at least 9amino acids of SEQ ID NO: 2, at least 9 amino acids of SEQ ID NO: 4, orat least 9 amino acids of SEQ ID NO: 46. The composition as described inthis paragraph may further comprise an effective amount of an immunestimulating agent. The present invention also provides that thecomposition as described in this paragraph is effective as a vaccine.

Further provided by the present invention is an assay kit for detectingantibodies against Leptospira strains which contains at least oneimmunologically active purified protein derived from Leptospirainterrogans wherein such protein is characterized in that it elicits animmunological response from a mammal, has been prepared by expression ina bacterium other than Leptospira interrogans, is free of otherLeptospira interrogans proteins; and is a protein having SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 46, at least 9 amino acids of SEQ ID NO: 2, atleast 9 amino acids of SEQ ID NO: 4, or at least 9 amino acids of SEQ IDNO: 46, which protein reacts with the antibodies present in a testfluid, and wherein said kit has at least one indicator component whichdetects complexes of immunologically active protein and antibody. Theindicator component of the assay kit of the present invention is anantibody which is directed against the antibody to be detected and whichhas a label. The assay kit of the present invention contains a label,which comprises a radioactive isotope, or an enzyme, such as peroxidase,which is able to catalyze a color or light reaction.

The present invention further provides that the immunologically activeprotein in the assay kit is biotinylated, and the indicator component isavidin or streptavidin having an enzyme covalently bonded thereto. Thepresent invention provides an ELISA assay kit. The present inventionprovides an assay kit as described hereinabove, wherein the at least oneimmunologically active protein is coupled to microtiter plates, and theindicator component comprises anti-human immunoglobulin to which anenzyme catalyzing a color reaction is coupled. The present inventionalso provides an assay kit as described hereinabove, wherein theindicator component comprises IgG antibodies, IgM antibodies or amixture thereof.

Provided by the present invention is a method for the detection ofLeptospira infection, comprising the steps of contacting LigA proteinfrom Leptospira interrogans with a biological sample from a mammalsuspected of having Leptospira infection, wherein the LigA protein ischaracterized in that it is a protein having SEQ ID NO: 2, or at least 9amino acids of SEQ ID NO: 2, and detecting the presence or absence of acomplex formed between LigA and antibodies in the biological sample.

Further provided by the present invention is a method for the detectionof Leptospira infection, comprising the steps of contacting LigB proteinfrom Leptospira interrogans with a biological sample from a mammalsuspected of having Leptospira infection, wherein the LigB protein ischaracterized in that it is a protein having SEQ ID NO: 4, SEQ ID NO:46, at least 9 amino acids of SEQ ID NO: 4, or at least 9 amino acids ofSEQ ID NO: 46, and detecting the presence or absence of a complex formedbetween LigB and antibodies in the biological sample.

Additionally provided by the present invention is an antibody specificfor the purified LigA polypeptide and the purified LigB polypeptide, asdescribed hereinabove. The present invention further provides formonoclonal or polyclonal antibodies, and the methods of makingmonoclonal or polyclonal antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequence of ligA (SEQ ID NO: 1) and its deduced aminoacid sequence (SEQ ID NO: 2). Bold regions are the three possibletranslation start codons. Underlined nucleotides indicate primerannealing sites for FIGS. 3 and 7, respectively. Arrows show thepotential transcription termination sequence.

FIG. 2. Alignment of the predicted amino acid sequences for the twelvetandem repeats (SEQ ID NOs: 5-16) and the immunoglobulin-like domain ofE. coli intimin binding (receptor) protein (Ig11, CD: pfam02368 (SEQ IDNO: 17); Ig12, CD: smart00635 (SEQ ID NO: 18)). Twelve repeat sequencesof a 90 amino acid sequence are from 136-218, 224-310, 311-400, 401-489,490-580, 581-670, 671-760, 761-851, 852-942, 943-1033, 1034-1125 and1126-1216, respectively.

FIG. 3. Expression of LigA in E. coli. Whole cell lysates of E. coliwere subjected to SDS-PAGE, transferred to nitrocellulose and blottedwith a 1:100 dilution of rabbit antiserum to the 90 kDa truncated LigA.Lanes 1 & 2. E. coli with vector, pET22b only. Lanes 3 & 4. E. coliharboring pET22b plus ligA construct. Lanes 2 & 4. E. coli was inducedwith 0.4 mM IPTG. Lane 5. Pre-stained molecular size markers (Bio-Rad,CA).

FIG. 4. LipL32 and LipL36 but not LigA expression are temperatureregulated. Lane 1. Whole cell lysate of leptospires grown at 30° C.Lanes 2, 3, 4, 5 and 6 represent 2, 3, 4, 5 and 6 day old cultures,respectively, of leptospires grown at 37° C. Each lane was loaded with˜5.0 μg of proteins.

FIG. 5. LigA expression in hamsters infected with L. interrogans serovarpomona. Sections of kidney were treated with rabbit antiserum specificfor a 90 kDa truncated LigA (A) L. interrogans serovar pomona (B),LipL32 (C), LipL36 (D) and with pre-immune serum (E). Kidney sectionsfrom non-infected hamsters were unreactive. Bar=67 μm.

FIG. 6. Recombinant LigA protein purified using metal affinitychromatography and subjected to SDS-PAGE separation was probed withnormal horse sera (first 4 lanes), equine lyme disease positive sera(lanes 5-9), human granulocytic ehrlichiosis positive sera (lanes10-11), aborted mare sera (lanes 12-19), and rabbit serum specific for a90 kDa truncated LigA (lane 20). Each lane was loaded with ˜0.5 μg ofprotein.

FIG. 7. Agarose gel showing PCR products and restriction analysis ofligA from different pathogenic serovars of Leptospira (A) PCR productsof ligA (B) HindIII digested PCR product of ligA. Lane 1. L. interrogansserovar pomona type kennewicki, 2. L. interrogans serovar pomona, 3. L.interrogans serovar hardjo, 4. L. interrogans serovaricterohemorrhagiae, 5. L. kirchneri serovar grippotyphosa, 6. L.interrogans serovar wolfii.

FIG. 8. Nucleotide sequences of the ligB (SEQ ID NO:3) and its deducedamino acid sequence (SEQ ID NO:4). Bold represents three possible startcodons and Italics indicate the potential ribosome-binding site. Thepredicted signal sequence of LigB is underlined. Serine rich region anda possible tyrosine kinase phosphorylation are earmarked in dotted linesand double underlined. Waveline indicates the homology region with IBDof Tir-intimin complex. The Genbank accession number for the nucleotidesequence of ligB is AF368236.

FIG. 9. Comparison of the structural domains of LigB with LigA from L.interrogans serovar pomona invasin from Yersinia and intimin from E.coli. Dark and dotted boxes represent the Ig-like domain and C typelectin like domain. LysM represents lysing motif in E. coli. ORFU showsthe open reading frame present in Tir and Intimin of E. coli.

FIG. 10. Alignment of twelve repeats of 90 amino acid sequence of LigB(SEQ ID NOs: 19-30) and its homology with the bacterial Ig-like domainfrom Pfam ((Ig1 (SEQ ID NO:31) and Ig2 (SEQ ID NO:32)). Gaps have beenintroduced to optimize alignment among the polypeptides.

FIG. 11. (A) Alignment of C-terminal variation regions of LigA (SEQ IDNO: 33) and LigB (SEQ ID NO: 34).

FIG. 12. Nucleotide sequence of ligB (SEQ ID NO: 45) and its deducedamino acid sequence (SEQ ID NO: 46).

FIG. 13. A. Alignment of twelve repeats of 90 amino acid sequence ofLigB indicate homology with the bacterial Ig-like domain from Pfam (Ig1and Ig2). Gaps have been introduced to optimize alignment among thepolypeptides. B. Alignment of variable regions of LigA and LigB.

FIG. 14. The presence of lig genes in different serovars of Leptospirawas determined by Souther blot. The non-radioactively labeled conservedregion of LigA and LigB were used to probe EcoRI digested genomic DNAfrom Leptospira interrogans serovars Pomona (Lane 1), Hardjo (Lane 2),Copenhageni (Lane 3), Grippotyphosa (Lane 4), Canicola (Lane 5), Wolffi(Lane 6), Autumnalis (Lane 7), Bataviae (Lane 8), Australis (Lane 9),and Pyrogenes (Lane 10). Non-pathogenic L. biflexa serovar Patoc doesnot contain the lig genes (Lane 11).

FIG. 15. Expression and purification of GST fusion proteins of LigA andLigB. LigA and LigB were truncated into conserved and variable regionscloned into plasmid pGEX4T-2 and expressed as GST fusion proteins. Thefusion proteins were purified by affinity chromatography and subjectedto SDS-PAGE. Expression and purification of the conserved regions ofLigA and LigB (A); expression of Variable region of LigA (B); expressionof Variable region of LigB (C). Lane 1. Molecular Marker (Bio-Rad, CA);lane 2. E. coli with vector, pGEX4T-2 only (control); lane 3, un-inducedE. coli with recombinant construct; lane 4, IPTG induced E. coli withrecombinant construct; lane 5, Affinity chromatography purified GSTfusion proteins; lane 6, Thrombin digested GST fusion protein.

FIG. 16. Immunoblot showing lack of expression of LigA and LigB inleptospires. Whole cell lysates of low passage and high passage culturesof leptospires, purified recombinant GST fusion proteins and thrombindigested GST fusion protein were then subjected to SDS-PAGE andtransferred to a nitrocellulose membrane. Membranes were then blottedwith a 1:800 dilution of a rabbit antiserum to the variable region ofLigA and LigB. 16A and 16B represent the expression of LigA and LigBrespectively. Lane 1, E. coli with vector; lanes 2 and 4, uninduced andIPTG induced E. coli harboring recombinant construct; lane 3, Thrombindigested, purified GST fusion protein; lanes 5 and 6, Low passage andhigh passage leptospires; lane 7, pre-stained molecular marker (Bio-Rad,CA).

FIG. 17. Expression of LigA and LigB of leptospires at the transcriptlevel. RNA from low and high passage cultures were subjected to one stepRT-PCR with ligA and ligB specific primers. FIGS. 17A and B representRT-PCR with a ligA and a ligB specific primer, respectively; Lane 1,Marker (pBH20 digested with HinfI); lanes 2 and 3, RNA from Low and highpassage cultures; lane 4, genomic DNA from the leptospires (positivecontrol); lane 5, control (RNA without RT in the reaction).

FIG. 18. Reactivity of vaccinated sera to whole cell proteins ofleptospires. Whole cell proteins of leptospires were subjected toSDS-PAGE, transferred to nitrocellulose membrane and probed with a 1:10dilution of pre- and post-vaccinated sera. Lane 1. Pre-vaccinated sera;lane 2. Grippo/pomona Vaccinated sera; lanes 4 and 5. Naturally infectedsera from dogs.

FIG. 19. KELA with recombinant antigens of LigA and LigB to MAT positivecanine sera. FIGS. 19A, B and C represent the reactivity of KELA usingrecombinant proteins from conserved regions of LigA and LigB (Con),variable region of LigA (VarA) and variable region of LigB (VarB) to MATpositive canine sera respectively. ♦Represents the reactivity of eachsample. Descriptive statistics was used to determine the cut off valuefor KELA units and the maximum reactivity of sera from healthy dogs wasconsidered as the cut off value (KELA cut off value for Con, VarA andVarB was 7, 42 and 42 respectively).

DETAILED DESCRIPTION OF THE INVENTION

I. Leptospira

Leptospira organisms are very thin, tightly coiled, obligate aerobicspirochetes characterized by a unique flexuous type of motility.Leptospira is a gram-negative spirochete with internal flagella. Thegenus is divided into two species: the pathogenic leptospires L.interrogans and the free-living leptospire L. biflexa. Serotypes of L.interrogans are the agents of leptospirosis, a zoonotic disease.

Leptospira enters the host through mucosa and broken skin, resulting inbacteremia. The spirochetes multiply in organs, most commonly thecentral nervous system, kidneys, and liver. They are cleared by theimmune response from the blood and most tissues but persist and multiplyfor some time in the kidney tubules. Infective bacteria are shed in theurine. The mechanism of tissue damage is not known.

The primary hosts for this disease are wild and domestic animals, andthe disease is a major cause of economic loss in the meat and dairyindustry. Humans are accidental hosts in whom this disseminated diseasevaries in severity from subclinical to fatal. Humans acquire theinfection by contact with the urine of infected animals. Human-to-humantransmission is very rare. The first human case of leptospirosis wasdescribed in 1886 as a severe icteric illness and was referred to asWeil's disease; however, most human cases of leptospirosis arenonicteric and are not life-threatening. Recovery usually follows theappearance of a specific antibody.

Clinical diagnosis is usually confirmed by serology. Isolation ofspirochetes is possible, but it is time-consuming and requires specialmedia. Serum antibodies are responsible for host resistance.

Clinical manifestations of leptospirosis are associated with a generalfebrile disease and are not sufficiently characteristic for diagnosis.As a result, leptospirosis often is initially misdiagnosed as meningitisor hepatitis. Typically, the disease is biphasic, which an acuteleptospiremic phase followed by the immune leptospiruric phase. Thethree organ systems most frequently involved are the central nervoussystem, kidneys, and liver. After an average incubation period of 7 to14 days, the leptospiremic acute phase is evidenced by abrupt onset offever, severe headache, muscle pain, and nausea; these symptoms persistfor approximately 7 days. Jaundice occurs during this phase in moresevere infections. With the appearance of antileptospiral antibodies,the acute phase of the disease subsides and leptospires can no longer beisolated from the blood. The immune leptospiruric phase occurs after anasymptomatic period of several days. It is manifested by a fever ofshorter duration and central nervous system involvement (meningitis).Leptospires appear in the urine during this phase and are shed forvarious periods depending on the host.

Leptospira has the general structural characteristics that distinguishspirochetes from other bacteria. The cell is encased in a three- tofive-layer outer membrane or envelope. Beneath this outer membrane arethe flexible, helical peptidoglycan layer and the cytoplasmic membrane;these encompass the cytoplasmic contents of the cell. The structuressurrounded by the outer membrane are collectively called theprotoplasmic cylinder. An unusual feature of the spirochetes is thelocation of the flagella, which lie between the outer membrane and thepeptidoglycan layer. They are referred to as periplasmic flagella. Theperiplasmic flagella are attached to the protoplasmic cylindersubterminally at each end and extend toward the center of the cell. Thenumber of periplasmic flagella per cell varies among the spirochetes.The motility of bacteria with external flagella is impeded in viscousenvironments, but that of spirochetes is enhanced. The slender (0.1 μmby 8 to 20 μm) leptospires are tightly coiled, flexible cells. In liquidmedia, one or both ends are usually hooked. Leptospires are too slenderto be visualized with the bright-field microscope, but are clearly seenby dark-field or phase microscopy. They do not stain well with anilinedyes.

The leptospires have two periplasmic flagella, one originating at eachend of the cell. The free ends of the periplasmic flagella extend towardthe center of the cell, but do not overlap as they do in otherspirochetes. The basal bodies of Leptospira periplasmic flagellaresemble those of Gram-negative bacteria, whereas those of otherspirochetes are similar to the basal bodies of Gram-positive bacteria.Leptospira differs from other spirochetes in that they are lackinglycolipids and having diaminopimelic acid rather than ornithine in itspeptidoglycan.

The leptospires are the most readily cultivated of the pathogenicspirochetes. They have relatively simple nutritional requirements;long-chain fatty acids and vitamins B1 and B12 are the only organiccompounds known to be necessary for growth. When cultivated in media ofpH 7.4 at 30° C., their average generation time is about 12 hours.Aeration is required for maximal growth. They can be cultivated inplates containing soft (1 percent) agar medium, in which they formprimarily subsurface colonies.

The two species, L. interrogans and L. biflexa, are further divided intoserotypes based on their antigenic composition. More than 200 serotypeshave been identified in L. interrogans. The most prevalent serotypes inthe United States are canicola, grippotyphosa, hardjo,icterohaemorrhagiae, and pomona. Genetic studies have demonstrated thatserologically diverse serotypes may be present in the same geneticgroup. At least seven species of pathogenic leptospires have beenidentified by nucleotide analysis.

The mucosa and broken skin are the most likely sites of entry for thepathogenic leptospires. A generalized infection ensues, but no lesiondevelops at the site of entry. Bacteremia occurs during the acute,leptospiremic phase of the disease. The host responds by producingantibodies that, in combination with complement, are leptospiricidal.The leptospires are rapidly eliminated from all host tissues except thebrain, eyes, and kidneys. Leptospires surviving in the brain and eyesmultiply slowly if at all; however, in the kidneys they multiply in theconvoluted tubules and are shed in the urine (the leptospiruric phase).The leptospires may persist in the host for weeks to months; in rodentsthey may be shed in the urine for the lifetime of the animal.Leptospiruric urine is the vehicle of transmission of this disease.

The mechanism by which leptospires cause disease remains unresolved, asneither endotoxins nor exotoxins have been associated with them. Themarked contrast between the extent of functional impairment inleptospirosis and the scarcity of histologic lesions suggests that mostdamage occurs at the subcellular level. Damage to the endothelial liningof the capillaries and subsequent interference with blood flow appearresponsible for the lesions associated with leptospirosis. The mostnotable feature of severe leptospirosis is the progressive impairment ofhepatic and renal function. Renal failure is the most common cause ofdeath. The lack of substantial cell destruction in leptospirosis isreflected in the complete recovery of hepatic and renal function insurvivors. Although spontaneous abortion is common in infected cattleand swine, only recently has a human case of fatal congenitalleptospirosis been documented.

The host's immunologic response to leptospirosis is though to beresponsible for lesions associated with the late phase of this disease;this helps to explain the ineffectiveness of antibiotics once symptomsof the disease have been present for 4 days or more.

Nonspecific host defenses appear ineffective against the virulentleptospires, which are rapidly killed in vitro by theantibody-complement system; virulent strains are more resistant to thisleptospiricidal activity than are avirulent strains. Immunity toleptospirosis is primarily humoral; cell-mediated immunity does notappear to be important, but may be responsible for some of the latemanifestations of the disease. Immunity to leptospirosis is serotypespecific and may persist for years. Immune serum has been used to treathuman leptospirosis and passively protects experimental animals from thedisease. The survival of leptospires with in the convoluted tubules ofthe kidneys may be related to the ineffectiveness of theantibody-complement system at this site. Previously infected animals canbecome seronegative and continue to shed leptospires in their urine,possibly because of the lack of antigenic stimulation by leptospires inthe kidneys.

Because clinical manifestations of leptospirosis are too variable andnonspecific to be diagnostically useful, microscopic demonstration ofthe organisms, serologic tests, or both are used in diagnosis. Themicroscopic agglutination test is most frequently used forserodiagnosis. The organisms can be isolated from blood or urine oncommercially available media, but the test must be requestedspecifically because special media is needed. Isolation of the organismsconfirms the diagnosis.

Reducing its prevalence in wild and domestic animals can control humanleptospirosis. Although little can be done about controlling the diseasein wild animals, leptospirosis in domestic animals can be controlledthrough vaccination with inactivated whole cells or an outer membranepreparation. If vaccines do not contain a sufficient immunogenic mass,the resulting immune response protects the host against clinicaldisease, but not against development of the renal shedder state. Becausea multiplicity of serotypes may exist in a given geographic region andthe protection afforded by the inactivated vaccines is serotypespecific, the use of polyvalent vaccines is usually recommended.

Although the leptospires are susceptible to antibiotics such aspenicillin and tetracycline in vitro, use of these drugs in thetreatment of leptospirosis is somewhat controversial. Treatment is mosteffective if initiated within a week of disease onset. At later times,immunologic damage may already have begun, rendering antimicrobialtherapy less effective. Doxycycline has been used successfully as achemoprophylactic agent for military personnel training in tropicalareas.

II. LigA and LigB

The ligA (SEQ ID NO: 1) and ligB (SEQ ID NO:3, SEQ ID NO:45) encodeLeptospiral immunoglobin (Ig)-like protein A and B (LigA and LigB) fromLeptospira interrogans serovar pomona type kennewicki and have molecularmasses of approximately 130 and 140 kDa, respectively.

LigA has twelve or more repeats of a 90 amino acid motif homologous withbacterial Ig-like domains such as intimins of E. coli, invasin ofYersinia and a cell surface protein of C. acetobutylicum (Palaniappan,R. U. M. et al., Infect. Immun., 70:5924-5930 (2002)).

The complete nucleotide sequence of a novel ligB is homologous with ligAof Leptospira, and cell adhesion proteins such as intimin of E. coli anda cell adhesion protein of C. acetobutylicum.

III. Definitions

The term “chimeric” refers to any gene or DNA that contains 1) DNAsequences, including regulatory and coding sequences, that are not foundtogether in nature, or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orcomprise regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different from that found in nature.

“Expression” refers to the transcription and/or translation of anendogenous gene or a transgene in a host cell. For example, in the caseof antisense constructs, expression may refer to the transcription ofthe antisense DNA only. In addition, expression refers to thetranscription and stable accumulation of sense (mRNA) or functional RNA.Expression may also refer to the production of protein.

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNA,or specific protein, including regulatory sequences. Genes also includenonexpressed DNA segments that, for example, form recognition sequencesfor other proteins. Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, DNA that is either heterologous or homologous to the DNA of aparticular cell to be transformed. Additionally, transgenes may comprisenative genes inserted into a non-native organism, or chimeric genes. Theterm “endogenous gene” refers to a native gene in its natural locationin the genome of an organism. A “foreign” or “exogenous” gene refers toa gene not normally found in the host organism but that is introduced bygene transfer.

The invention encompasses isolated or substantially purified nucleicacid compositions. In the context of the present invention, for example,an “isolated” or “purified” DNA molecule is a DNA molecule that existsapart from its native environment and is therefore not a product ofnature. An isolated DNA molecule may exist in a purified form or mayexist in a non-native environment such as, for example, a transgenichost cell. For example, an “isolated” or “purified” nucleic acidmolecule, or biologically active portion thereof, is substantially freeof other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. In one embodiment, an“isolated” nucleic acid is free of sequences that naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Fragments and variants of the disclosednucleotide sequences and proteins or partial-length proteins encodedthereby are also encompassed by the present invention. By “fragment” or“portion” is meant a full length, or less than full length, of thenucleotide sequence encoding, or the amino acid sequence of, apolypeptide or protein.

A “mutation” refers to an insertion, deletion or substitution of one ormore nucleotide bases of a nucleic acid sequence, so that the nucleicacid sequence differs from the wild-type sequence. For example, a“point” mutation refers to an alteration in the sequence of a nucleotideat a single base position from the wild type sequence.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991);Ohtsuka et al., JBC, 260:2605 (1985); Rossolini et al., Mol. Cell.Probes, 8:91 (1994). A “nucleic acid fragment” is a fraction of a givennucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority oforganisms is the genetic material while ribonucleic acid (RNA) isinvolved in the transfer of information contained within DNA intoproteins. The term “nucleotide sequence” refers to a polymer of DNA orRNA that can be single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The terms “nucleic acid,”“nucleic acid molecule,” “nucleic acid fragment,” “nucleic acidsequence,” or “polynucleotide” may also be used interchangeably withgene, cDNA, DNA and RNA encoded by a gene (Batzer et al., 1991; Ohtsukaet al., 1985; Rossolini et al., 1999).

“Operably linked” when used with respect to nucleic acid, means joinedas part of the same nucleic acid molecule, suitably positioned andoriented for transcription to be initiated from the promoter. DNAoperably linked to a promoter is under transcriptional initiationregulation of the promoter. Coding sequences can be operably-linked toregulatory sequences in sense or antisense orientation.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of, for example, a TATA-box, a -35, -10polymerase binding site and/or a ribosome binding site (Shine-Dolgarnosequence), and other sequences that serve to specify the site oftranscription initiation, to which regulatory elements are added forcontrol of expression. “Promoter” also refers to a nucleotide sequencethat includes a minimal promoter plus regulatory elements that iscapable of controlling the expression of a coding sequence or functionalRNA. This type of promoter sequence consists of proximal and more distalupstream elements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. It is capable of operating in both orientations (normal orflipped), and is capable of functioning even when moved either upstreamor downstream from the promoter. Both enhancers and other upstreampromoter elements bind sequence-specific DNA-binding proteins thatmediate their effects. Promoters may be derived in their entirety from anative gene, or be composed of different elements derived from differentpromoters found in nature, or even be comprised of synthetic DNAsegments. A promoter may also contain DNA sequences that are involved inthe binding of protein factors that control the effectiveness oftranscription initiation in response to physiological or developmentalconditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, a -35, -10 polymerasebinding site and/or a ribosome binding site (Shine-Dolgarno sequence),that are inactive or that have greatly reduced promoter activity in theabsence of upstream activation are referred to as “minimal or corepromoters.” In the presence of a suitable transcription factor, theminimal promoter functions to permit transcription. A “minimal or corepromoter” thus consists only of all basal elements needed fortranscription initiation, e.g., a TATA box, a -35, -10 polymerasebinding site, a ribosome binding site (Shine-Dolgarno sequence), and/oran initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter. An “inducible promoter”is a regulated promoter that can be turned on in a cell by an externalstimulus, such as a chemical, light, hormone, stress, or a pathogen.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

-   -   (a) As used herein, “reference sequence” is a defined sequence        used as a basis for sequence comparison. A reference sequence        may be a subset or the entirety of a specified sequence; for        example, as a segment of a full-length cDNA or gene sequence, or        the complete cDNA or gene sequence.    -   (b) As used herein, “comparison window” makes reference to a        contiguous and specified segment of a polynucleotide sequence,        wherein the polynucleotide sequence in the comparison window may        comprise additions or deletions (i.e., gaps) compared to the        reference sequence (which does not comprise additions or        deletions) for optimal alignment of the two sequences.        Generally, the comparison window is at least 20 contiguous        nucleotides in length, and optionally can be 30, 40, 50, 100, or        longer. Those of skill in the art understand that to avoid a        high similarity to a reference sequence due to inclusion of gaps        in the polynucleotide sequence a gap penalty is typically        introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, CABIOS, 4:11 (1988); the local homology algorithmof Smith et al., Adv. Appl. Math., 2:482 (1981); the homology alignmentalgorithm of Needleman and Wunsch, JMB, 48:443 (1970); thesearch-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad.Sci. USA, 85:2444 (1988); the algorithm of Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 87:2264 (1990), modified as in Karlin andAltschul, Proc. Natl. Acad. Sci. USA, 90:5873 (1993).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.,Gene, 73:237 (1988); Higgins et al., CABIOS, 5:151 (1989); Corpet etal., Nucl. Acids Res., 16:10881 (1988); Huang et al., CABIOS, 8:155(1992); and Pearson et al., Meth. Mol. Biol., 24:307 (1994). The ALIGNprogram is based on the algorithm of Myers and Miller, supra. The BLASTprograms of Altschul et al., JMB, 215:403 (1990); Nucl. Acids Res.,25:3389 (1990), are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information, which is available on theworld wide web at ncbi.nlm.nih.gov. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al., NucleicAcids Res. 25:3389 (1997). Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al., supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g. BLASTN for nucleotide sequences, BLASTX for proteins) canbe used. The BLASTN program (for nucleotide sequences) uses as defaultsa wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5,N=−4, and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix. See the world wide web atncbi.n1m.nih.gov. Alignment may also be performed manually byinspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

-   -   (c) As used herein, “sequence identity” or “identity” in the        context of two nucleic acid or polypeptide sequences makes        reference to a specified percentage of residues in the two        sequences that are the same when aligned for maximum        correspondence over a specified comparison window, as measured        by sequence comparison algorithms or by visual inspection. When        percentage of sequence identity is used in reference to proteins        it is recognized that residue positions which are not identical        often differ by conservative amino acid substitutions, where        amino acid residues are substituted for other amino acid        residues with similar chemical properties (e.g., charge or        hydrophobicity) and therefore do not change the functional        properties of the molecule. When sequences differ in        conservative substitutions, the percent sequence identity may be        adjusted upwards to correct for the conservative nature of the        substitution. Sequences that differ by such conservative        substitutions are said to have “sequence similarity” or        “similarity.” Means for making this adjustment are well known to        those of skill in the art. Typically this involves scoring a        conservative substitution as a partial rather than a full        mismatch, thereby increasing the percentage sequence identity.        Thus, for example, where an identical amino acid is given a        score of 1 and a non-conservative substitution is given a score        of zero, a conservative substitution is given a score between        zero and 1. The scoring of conservative substitutions is        calculated, e.g., as implemented in the program PC/GENE        (Intelligenetics, Mountain View, Calif.).    -   (d) As used herein, “percentage of sequence identity” means the        value determined by comparing two optimally aligned sequences        over a comparison window, wherein the portion of the        polynucleotide sequence in the comparison window may comprise        additions or deletions (i.e., gaps) as compared to the reference        sequence (which does not comprise additions or deletions) for        optimal alignment of the two sequences. The percentage is        calculated by determining the number of positions at which the        identical nucleic acid base or amino acid residue occurs in both        sequences to yield the number of matched positions, dividing the        number of matched positions by the total number of positions in        the window of comparison, and multiplying the result by 100 to        yield the percentage of sequence identity.    -   (e)(i) The term “substantial identity” of polynucleotide        sequences means that a polynucleotide comprises a sequence that        has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or        79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,        88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or        94%, and most preferably at least 95%, 96%, 97%, 98%, or 99%        sequence identity, compared to a reference sequence using one of        the alignment programs described using standard parameters. One        of skill in the art will recognize that these values can be        appropriately adjusted to determine corresponding identity of        proteins encoded by two nucleotide sequences by taking into        account codon degeneracy, amino acid similarity, reading frame        positioning, and the like. Substantial identity of amino acid        sequences for these purposes normally means sequence identity of        at least 70%, more preferably at least 80%, 90%, and most        preferably at least 95%. Another indication that nucleotide        sequences are substantially identical is if two molecules        hybridize to each other under stringent conditions (see below).        Generally, stringent conditions are selected to be about 5° C.        lower than the thermal melting point (T_(m)) for the specific        sequence at a defined ionic strength and pH. However, stringent        conditions encompass temperatures in the range of about 1 ° C.        to about 20° C., depending upon the desired degree of stringency        as otherwise qualified herein. Nucleic acids that do not        hybridize to each other under stringent conditions are still        substantially identical if the polypeptides they encode are        substantially identical. This may occur, e.g., when a copy of a        nucleic acid is created using the maximum codon degeneracy        permitted by the genetic code. One indication that two nucleic        acid sequences are substantially identical is when the        polypeptide encoded by the first nucleic acid is immunologically        cross reactive with the polypeptide encoded by the second        nucleic acid.    -   (e)(ii) The term “substantial identity” in the context of a        peptide indicates that a peptide comprises a sequence with at        least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%,        preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%,        more preferably at least 90%, 91%, 92%, 93%, or 94%, or even        more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to        the reference sequence over a specified comparison window.        Preferably, optimal alignment is conducted using the homology        alignment algorithm of Needleman and Wunsch, J. Mol. Biol.        48:443 (1970). An indication that two peptide sequences are        substantially identical is that one peptide is immunologically        reactive with antibodies raised against the second peptide.        Thus, a peptide is substantially identical to a second peptide,        for example, where the two peptides differ only by a        conservative substitution.    -   For sequence comparison, typically one sequence acts as a        reference sequence to which test sequences are compared. When        using a sequence comparison algorithm, test and reference        sequences are input into a computer, subsequence coordinates are        designated if necessary, and sequence algorithm program        parameters are designated. The sequence comparison algorithm        then calculates the percent sequence identity for the test        sequence(s) relative to the reference sequence, based on the        designated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The thermal melting point(T_(m)) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl, Anal. Biochem.,138:267 (1984); T_(m) 81.5° C. +16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, %GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. T_(m) is reduced by about 1° C. foreach 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the T_(m) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lower than the T_(m); moderately stringent conditionscan utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lowerthan the T_(m); low stringency conditions can utilize a hybridizationand/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the T_(m). Usingthe equation, hybridization and wash compositions, and desiredtemperature, those of ordinary skill will understand that variations inthe stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a temperatureof less than 45° C. (aqueous solution) or 32° C. (formamide solution),it is preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology Hybridization with Nucleic Acid Probes, part Ichapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays” Elsevier, N.Y. (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the T_(m) for the specific sequence at a defined ionicstrength and pH.

An example of highly stringent wash conditions is 0. 1 5 M NaCl at 72°C. for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M, more preferably about 0.01 to 1.0 M, Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. and at least about 60° C. for long probes (e.g., >50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Nucleic acids that do not hybridize to eachother under stringent conditions are still substantially identical ifthe proteins that they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein. The term protein, as used herein, generallyrefers to a long, linear polymer of amino acids joined head to tail by apeptide bond between the carboxylic acid group of one amino acid and theamino group of the next.

As used herein, the term “immunogenic protein” refers to a protein thatis capable of inducing a humoral and/or a cell-mediated immune response.A substance that induces a specific immune response may also be referredto as an “antigen,” an “immunogen,” or an “immunologically activeprotein.”

As used herein, the term “leptospiral protein” includes variants orbiologically active or inactive fragments of LigA or LigB fromLeptospira interrogans. A “variant” of the polypeptide is a leptospiralprotein that is not completely identical to a native leptospiralprotein. A variant leptospiral protein can be obtained by altering theamino acid sequence by insertion, deletion, or substitution of one ormore amino acid. The amino acid sequence of the protein is modified, forexample by substitution, to create a polypeptide having substantiallythe same or improved qualities as compared to the native polypeptide.The substitution may be a conserved substitution. A “conservedsubstitution” is a substitution of an amino acid with another amino acidhaving a similar side chain. A conserved substitution would be asubstitution with an amino acid that makes the smallest change possiblein the charge of the amino acid or size of the side chain of the aminoacid (alternatively, in the size, charge or kind of chemical groupwithin the side chain) such that the overall peptide retains its spatialconformation but has altered biological activity. For example, commonconserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg orPhe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine iscommonly used to substitute for other amino acids. The 20 essentialamino acids can be grouped as follows: alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan and methionine havingnonpolar side chains; glycine, serine, threonine, cystine, tyrosine,asparagine and glutamine having uncharged polar side chains; aspartateand glutamate having acidic side chains; and lysine, arginine, andhistidine having basic side chains. Stryer, L. Biochemistry (2d edition)W. H. Freeman and Co. San Francisco (1981), p. 14-15; Lehninger, A.Biochemistry (2d ed., 1975), p. 73-75.

It is known that variant polypeptides can be obtained based onsubstituting certain amino acids for other amino acids in thepolypeptide structure in order to modify or improve biological activity.For example, through substitution of alternative amino acids, smallconformational changes may be conferred upon a polypeptide that resultin increased bioactivity. Alternatively, amino acid substitutions incertain polypeptides may be used to provide residues that may then belinked to other molecules to provide peptide-molecule conjugates thatretain sufficient properties of the starting polypeptide to be usefulfor other purposes.

One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, wherein it is foundthat certain amino acids may be substituted for other amino acids havingsimilar hydropathic indices and still retain a similar biologicalactivity. Alternatively, substitution of like amino acids may be made onthe basis of hydrophilicity, particularly where the biological functiondesired in the polypeptide to be generated in intended for use inimmunological embodiments. The greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity. U.S. Pat. No. 4,554,101.Accordingly, it is noted that substitutions can be made based on thehydrophilicity assigned to each amino acid. In using either thehydrophilicity index or hydropathic index, which assigns values to eachamino acid, it is preferred to conduct substitutions of amino acidswhere these values are ±2, with ±1 being particularly preferred, andthose with in ±0.5 being the most preferred substitutions.

The variant leptospiral protein comprises at least seven amino acidresidues, preferably about 20 to about 2000 residues, and morepreferably about 50 to about 1000 residues, and even more preferablyabout 80 to about 200 residues, wherein the variant leptospiral proteinhas at least 50%, preferably at least about 80%, and more preferably atleast about 90% but less than 100%, contiguous amino acid sequencehomology or identity to the amino acid sequence of a correspondingnative leptospiral protein.

The amino acid sequence of the variant leptospiral protein correspondsessentially to the native leptospiral protein amino acid sequence. Asused herein “correspond essentially to” refers to a polypeptide sequencethat will elicit a protective immunological response substantially thesame as the response generated by native leptospiral protein. Such aresponse may be at least 60% of the level generated by nativeleptospiral protein, and may even be at least 80% of the level generatedby native leptospiral protein. An immunological response to acomposition or vaccine is the development in the host of a cellularand/or antibody-mediated immune response to the polypeptide or vaccineof interest. Usually, such a response consists of the subject producingantibodies, B cell, helper T cells, suppressor T cells, and/or cytotoxicT cells directed specifically to an antigen or antigens included in thecomposition or vaccine of interest.

A variant of the invention may include amino acid residues not presentin the corresponding native leptospiral protein, or may includedeletions relative to the corresponding native leptospiral protein. Avariant may also be a truncated “fragment” as compared to thecorresponding native leptospiral protein, i.e., only a portion of afull-length protein. Leptospiral protein variants also include peptideshaving at least one D-amino acid.

The immunologically active proteins of the present invention areleptospiral proteins, as well as proteins, polypeptides, variants orfragments of LigA or LigB from Leptospira interrogans. Theimmunologically active proteins of the present invention may be ofvariable length, with the minimum fragment length comprising between9-15 amino acids.

As used herein, a “transgenic,” “transformed,” or “recombinant” cellrefers to a genetically modified or genetically altered cell, the genomeof which comprises a recombinant DNA molecule or sequence (“transgene”).For example, a “transgenic cell” can be a cell transformed with a“vector.” A “transgenic,” “transformed,” or “recombinant” cell thusrefers to a host cell such as a bacterial or yeast cell into which aheterologous nucleic acid molecule has been introduced. The nucleic acidmolecule can be stably integrated into the genome by methods generallyknown in the art (e.g., disclosed in Sambrook and Russell, 2001). Forexample, “transformed,” “transformant,” and “transgenic” cells have beenthrough the transformation process and contain a foreign or exogenousgene. The term “untransformed” refers to cells that have not beenthrough the transformation process.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, or the transfer into a hostcell of a nucleic acid fragment that is maintained extrachromosomally.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor other construct in double or single stranded linear or circular formthat may or may not be self transmissible or mobilizable, and that cantransform prokaryotic or eukaryotic host either by integration into thecellular genome or exist extrachromosomally, e.g., autonomousreplicating plasmid with an origin of replication. A vector can comprisea construct such as an expression cassette having a DNA sequence capableof directing expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest that also is operably linked totermination signals. An expression cassette also typically comprisessequences required for proper translation of the nucleotide sequence.The expression cassette comprising the nucleotide sequence of interestmay be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.The expression of the nucleotide sequence in the expression cassette maybe under the control of a constitutive promoter or of an induciblepromoter that initiates transcription only when the host cell is exposedto some particular external stimulus.

The term “wild type” refers to an untransformed cell, i.e., one wherethe genome has not been altered by the presence of the recombinant DNAmolecule or sequence or by other means of mutagenesis. A “corresponding”untransformed cell is a typical control cell, i.e., one that has beensubjected to transformation conditions, but has not been exposed toexogenous DNA.

A “vaccine” is a compound or composition that will elicit a protectiveimmunological response in an animal to which the vaccine has beenadministered. An immunological response to a vaccine is the developmentin the host of a cellular and/or antibody-mediated immune response tothe polypeptide or vaccine of interest. Usually, such a responseconsists of the subject producing antibodies, B cell, helper T cells,suppressor T cells, and/or cytotoxic T cells directed specifically to anantigen or antigens included in the composition or vaccine of interest.

As used herein, the term “therapeutic agent” refers to any agent ormaterial that has a beneficial effect on the mammalian recipient. Thus,“therapeutic agent” embraces both therapeutic and prophylactic moleculeshaving nucleic acid or protein components.

IV. Vaccine Preparations

The present invention provides a vaccine for use to protect mammalsagainst Leptospira colonization or infection. For example, the vaccinemay contain an immunogenic amount of isolated and purified Leptospiraprotein or cell in combination with a physiologically-acceptable,non-toxic vehicle. Vaccines of the present invention can also includeeffective amounts of immunological adjuvants, known to enhance an immuneresponse.

To immunize a subject, the immunogenic protein from Leptospira isadministered parenterally, usually by intramuscular or subcutaneousinjection in an appropriate vehicle. Other modes of administration,however, are also acceptable. For example, the vaccine may beadministered orally, or via a mucosal route, such as a nasal,gastrointestinal or genital site. Vaccine formulations will contain aneffective amount of the active ingredient in a vehicle. The effectiveamount is sufficient to prevent, ameliorate or reduce the incidence ofLeptospira infection in the target mammal. The effective amount isreadily determined by one skilled in the art. The active ingredient maytypically range from about 1% to about 95% (w/w) of the composition, oreven higher or lower if appropriate. The quantity to be administereddepends upon factors such as the age, weight and physical condition ofthe animal considered for vaccination. The quantity also depends uponthe capacity of the animal's immune system to synthesize antibodies, andthe degree of protection desired. Effective dosages can be readilyestablished by one of ordinary skill in the art through routine trialsestablishing dose response curves. The subject is immunized byadministration of the leptospiral vaccine in one or more doses. Multipledoses may be administered as is required to maintain a state of immunityto Leptospira.

To prepare a vaccine, the immunogenic Leptospira protein or proteins canbe isolated, lyophilized and stabilized. The vaccine may then beadjusted to an appropriate concentration, optionally combined with asuitable vaccine adjuvant, and packaged for use. Suitable adjuvantsinclude but are not limited to surfactants, e.g., hexadecylamine,octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′-N-bis(2-hydroxyethyl-propane di-amine),methoxyhexadecyl-glycerol, and pluronic polyols; polanions, e.g., pyran,dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g.,muramyl dipeptide, MPL, aimethylglycine, tuftsin, oil emulsions, alum,and mixtures thereof. Other potential adjuvants include the B peptidesubunits of E. coli heat labile toxin or of the cholera toxin. (McGheeet al., 1993). Finally, the immunogenic product may be incorporated intoliposomes for use in a vaccine formulation, or may be conjugated toproteins such as keyhole limpet hemocyanin (KLH) or human serum albumin(HSA) or other polymers.

V. Antibodies

The antibodies of the invention are prepared by using standardtechniques. To prepare polyclonal antibodies or “antisera,” an animal isinoculated with an antigen, i.e., a purified immunogenic protein fromLeptospira, and then immunoglobulins are recovered from a fluid, such asblood serum, that contains the immunoglobulins, after the animal has hadan immune response.

For inoculation, the antigen is preferably bound to a carrier peptideand emulsified using a biologically suitable emulsifying agent, such asFreund's incomplete adjuvant. A variety of mammalian or avian hostorganisms may be used to prepare polyclonal antibodies againstLeptospira.

Following immunization, immunoglobulin is purified from the immunizedbird or mammal, e.g., goat, rabbit, mouse, rat, or donkey and the like.For certain applications, particularly certain pharmaceuticalapplications, it is preferable to obtain a composition in which theantibodies are essentially free of antibodies that do not react with theimmunogen. This composition is composed virtually entirely of the hightiter, monospecific, purified polyclonal antibodies to the Leptospiraprotein.

Antibodies can be purified by affinity chromatography, using purifiedLeptospira protein. Purification of antibodies by affinitychromatography is generally known to those skilled in the art (see, forexample, U.S. Pat. No. 4,533,630). Briefly, the purified antibody isbound to a solid support for a sufficient time and under appropriateconditions for the antibody to bind to the polypeptide or peptide. Suchtime and conditions are readily determinable by those skilled in theart. The unbound, unreacted antibody is then removed, such as bywashing. The bound antibody is then recovered from the column by elutingthe antibodies, so as to yield purified, monospecific polyclonalantibodies.

Monoclonal antibodies can be also prepared, using known hybridoma cellculture techniques. In general, this method involves preparing anantibody-producing fused cell line, e.g., of primary spleen cells fusedwith a compatible continuous line of myeloma cells, and growing thefused cells either in mass culture or in an animal species, such as amurine species, from which the myeloma cell line used was derived or iscompatible. Such antibodies offer many advantages in comparison to thoseproduced by inoculation of animals, as they are highly specific andsensitive and relatively “pure” immunochemically. Immunologically activefragments of the present antibodies are also within the scope of thepresent invention, e.g., the F_((ab)) fragment scFv antibodies, as arepartially humanized monoclonal antibodies.

Thus, it will be understood by those skilled in the art that thehybridomas herein referred to may be subject to genetic mutation orother changes while still retaining the ability to produce monoclonalantibody of the same desired specificity. The present inventionencompasses mutants, other derivatives and descendants of thehybridomas.

It will be further understood by those skilled in the art that amonoclonal antibody may be subjected to the techniques of recombinantDNA technology to produce other derivative antibodies, humanized orchimeric molecules or antibody fragments that retain the specificity ofthe original monoclonal antibody. Such techniques may involve combiningDNA encoding the immunoglobulin variable region, or the complementaritydetermining regions (CDRs), of the monoclonal antibody with DNA codingthe constant regions, or constant regions plus framework regions, of adifferent immunoglobulin, for example, to convert a mouse-derivedmonoclonal antibody into one having largely human immunoglobulincharacteristics (see EP 184187A, 2188638A, herein incorporated byreference).

VI. Formulations of Vaccine Compounds and Methods of Administration

The vaccine compounds may be formulated as pharmaceutical compositionsand administered to a mammalian host, such as a human patient, in avariety of forms adapted to the chosen route of administration, i.e.,orally or parenterally, by intravenous, intramuscular, topical orsubcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts may be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient that are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent, e.g., anadjuvant, with various of the other ingredients enumerated above, asrequired, followed by filter sterilization. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze dryingtechniques, which yield a powder of the active ingredient plus anyadditional desired ingredient present in the previously sterile-filteredsolutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions that can be used todeliver the compounds of the present invention to the skin are known tothe art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No.4,559,157)and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of the present inventionin a liquid composition, such as a lotion, will be from about 0.1-25wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1 -5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 10 ug/kg, e.g., from about 0.5 to about 10 ug/kg of bodyweight per day, such as 3 to about 5 ug per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 ug/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 10 to 50 ug, conveniently 15 to 50 ug, mostconveniently, about 50 ug of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

VII. Assay Kits

The present invention also includes a diagnostic kit for detecting ordetermining the presence of a Leptospira infection. The diagnostic kitmay also provide for the detection of the presence of LigA or LigB, or acombination thereof, in a physiological sample.

The kit comprises packaging, containing, separately packaged: (a) anamount of at least a first antibody which binds to a leptospiral proteinor polypeptide; and (b) instruction means. Preferably, the antibody islabeled or is bound by a detectable label or a second antibody that islabeled.

The immobilized antibodies to LigA or LigB, and labeled antibodies toLigA and LigB, may be conveniently packaged in kit form, wherein two ormore of the various immunoreagents will be separately packaged inpreselected amounts, within the outer packaging of the kit, which may bea box, envelope, or the like. The packaging also preferably comprisesinstruction means, such as a printed insert, a label, a tag, a cassettetape and the like, instructing the user in the practice of the assayformat.

For example, one such diagnostic kit for detecting or determining thepresence of LigA or LigB comprises packaging containing, separatelypackaged: (a) a solid surface, such as a fibrous test strip, amulti-well microliter plate, a test tube, or beads, having bound theretoantibodies to LigA or LigB; and (b) a known amount of antibodiesspecific to LigA or LigB, wherein said antibodies comprise a detectablelabel, or a binding site for a detectable label.

The kit may comprise a mixture of antibodies, each of which binds to adifferent epitope on the same leptospiral protein or polypeptide, e.g.,a mixture of antibodies comprising antibodies that bind to SEQ ID NO:2and antibodies that bind to SEQ ID NO:4, and/or SEQ ID NO:46.

The kit may also comprise a blocking agent, e.g., BSA, which may becontacted with a sample to be tested before contacting the sample withthe antibody, or may be contacted concurrently with the antibody. In oneembodiment of the invention, the kit further comprises a known amount ofa second antibody, which is detectably labeled or binds to a detectablelabel. The second antibody may bind to the same polypeptide as the firstantibody, or may bind to the first antibody. Preferably, the kit is adiagnostic kit.

Also provided is a kit useful to detect a leptospiral protein orpolypeptide in a sample. The kit comprises a solid substrate on whichthe sample to be tested is placed and a preparation of antibodies.Preferably, the antibodies are labeled or bind to a detectable label.

The invention will now be described by the following non-limitingexamples.

EXAMPLES Example I Identification of LigA

Characterization of bacterial antigens expressed only during infectionis essential in gaining a deeper understanding of infectious diseasessuch as leptospirosis. Immunoscreening of gene libraries withconvalescent serum is a powerful tool in the discovery of these in vivoexpressed immunogens, which would otherwise be difficult or impossibleto identify. It has been previously shown that sera from horses thataborted as a result of naturally acquired L. interrogans serovar pomonatype kennewicki infection recognize numerous periplasmic and outermembrane proteins, some of which are regulated by temperature (Nally, J.E. et al., Infect. Immun., 69:400-404 (2001)). In this study,immunoscreening of a genomic library of L. interrogans serovar pomonatype kennewicki was performed, and a novel, highly immunogenic proteinexpressed during equine infection (LigA) was identified.

Materials and Methods

Bacterial strains and culture conditions. L. interrogans serovar pomonatype kennewicki was provided by Dr. M. Donahue (Diagnostic Laboratory,Department of Veterinary Science, University of Kentucky) who isolatedthis strain from a case of ERU. Other serovars were obtained from theAmerican Type Culture Collection (ATCC) and maintained in the DiagnosticLaboratory at Cornell University. Leptospires were grown on PLM-5 medium(Intergen, NJ) at 30° C. Growth was monitored by dark field microscopy.Temperature regulation was carried out as previously described (Nally,J. E. et al., Infect. Immun., 69:400-404 (2001)).

Sera. Sera were obtained from mares that had recently aborted due toLeptospira infection. These sera had high titers for L. interrogansserovar pomona, as determined by the microscopic agglutination test. Inmost cases, the diagnosis was confirmed by microscopic detection ofleptospires in fetal tissues and the placenta. Rabbit anti-leptospiralantibody was obtained from NVSL, Iowa (1098-LEP-FAC). Antisera to LipL32and LipL36 were kindly provided by D. A. Haake (UCLA, CA).

Genomic DNA library. Genomic DNA was prepared from L. interrogansserovar pomona kennewicki as previously described (Chang, Y. F. et al.,DNA Cell. Biol., 12:351-362 (1993)). Partial restriction digestion wasperformed with TSP5091 and the digested fragments were ligated intopre-digested lambda Zap II (Stratagene). Ligated DNA was packaged intoGiga pack II Gold packaging extracts and stored in 0.3% chloroform.After transfection into E. coli MRF′ XL1 blue (Stratagene, CA), thelibrary was plated, amplified, and screened with convalescent mare'sserum according to the manufacturer's instructions (Stratagene, CA).

DNA sequencing and analysis. DNA sequencing was done using an ABI model377 automated nucleic acid sequencer at the Bioresource Center, CornellUniversity, NY. Homology searches were performed with NCBI, Blast(Altschul, S. F. et al., Nucleic Acid Res., 25:3389-3402 (1997)).Secondary structure, hydrophobicity and antigenic index predictions wereobtained by using PCgene and DNA star.

Expression of ligA in E. coli.

ligA without the signal sequence (deletion of the N-terminal 31 aminoacids) was amplified using primers (sense,5′-GGGTTTCATATGGCTGGCAAAAGAGGC-3′ (SEQ ID NO:35) and antisense,5′-CCCTCGAGTGGCTCCGTTTTAAT-3′ (SEQ ID NO:36)) and subcloned intoNdeI-XhoI sites of pET22b (Novagen, Madison, Wis.). The recombinantplasmid was transformed to E. coli BL21 (DE3) and expression was inducedby IPTG as previously described (Chang, Y.-F. et al., Vet. Parasitol.,78:137-145 (1998)).

A 90 kDa truncated LigA was subcloned into the XhoI-BamHI sites ofpET15b (Novagen) by PCR using primers (sense,5′-TCGAGGTCTCTCCAGTTTTACC-3′ (SEQ ID NO:37) and antisense,5′-GCGGATCCTGTTTTCATGTTATGGCTCC-3 ′)(SEQ ID NO:38). The resultingplasmid was transformed into E. coli BL21 (DE3) and the truncatedrecombinant LigA fusion protein was isolated from a lysate of BL21 byaffinity chromatography on His Bind Resin (Novagen).

Preparation of LigA specific rabbit antiserum. Antiserum to a 90 kDatruncated LigA was prepared in adult New Zealand rabbits. Recombinanttruncate was purified from periplasmic proteins of E. coli Nova bluethat contained pKS (Stratagene) encoding a 5 kb BamH1-Sal1 fragment orby affinity chromatography on Avidgel F (UniSyn Technology Inc., Tustin,Calif.) to which IgG from convalescent mare's serum had been coupled.The rabbits were immunized subcutaneously with 50 μg of the 90 kDatruncated LigA mixed with complete Freund's adjuvant on day 1 followedby a booster inoculum of 50 μg protein and incomplete Freund's adjuvanton days 10 and 19. On day 35, the rabbits were boosted intravenouslywith 50 μg of protein and then bled on day 45.

SDS PAGE and Immunoblot Analysis. Purified truncated LigA protein wassubjected to SDS-PAGE and immunoblot analysis as previously described(Chang, Y. F. et al., Infect. Immun., 63:3543-3549 (1995); Chang, Y. F.et al., DNA Cell. Biol., 12:351-362 (1993)). Rabbit antiserum totruncated LigA or convalescent mare's sera were used as primaryantibodies. Blots were developed with peroxidase conjugated protein G orgoat anti-horse IgG conjugated to alkaline phosphatase (KPL). Reactivebands were visualized by using 4-1 chloro-naphthol (0.5 mg/ml) ornitroblue tetrazolium and 5 bromo-3-chloro indolyl phosphate asappropriate.

Immunohistochemistry. Immunohistochemistry was performed on normal andleptospiral infected hamster kidneys usingbiotin-streptavidin-horseradish peroxidase according to themanufacturer's instructions (Zymed Laboratories, South San Francisco,Calif.). The chromagen was Nova Red (DAKO, Carpinteria, Calif.). Theprimary antibody was rabbit antiserum specific for truncated LigA andwas titrated by using a two-fold serial dilution from 1:10 to 1:320.Negative controls consisted of non-immune rabbit serum diluted 1:10,1:20 and 1:40. Anti-LipL32 was used as a positive control.

Kidneys were removed from leptospiral infected and normal hamsterseuthanized as part of an unrelated research project. These tissues wereimmediately embedded in O.C.T. Compound (Miles, Elkhart, Ind.) and snapfrozen in 2-methyl butane (Sigma, St. Louis, Mo.) prechilled to thepoint of freezing in liquid nitrogen.

Tissues were sectioned at 6 μm, mounted on Microscope Plus slides(Fisher Scientific), fixed in acetone for 2 minutes and air-dried.Endogenous peroxidase was quenched for 10 minutes in 0.3% hydrogenperoxide in 0. 1% w/v sodium azide and rinsed for 3 minutes in 0.01Mphosphate buffered saline, pH 7.6 (PBS). Sections were then blocked with10% heat inactivated goat serum for 10 minutes. The blocking serum wastipped off and the primary antibody applied for 60 minutes at roomtemperature. After rinsing 3 times in PBS, a 1:400 dilution ofbiotinylated goat anti-rabbit IgG was added for 20 minutes. Sectionswere rinsed 3 times and then incubated with a 1:400 dilution of thestreptavidin-peroxidase reagent for 10 minutes. After rinsing, thechromogen-substrate mixture was added to the sections and the reactionmonitored under the microscope until well developed or until backgrounddeveloped. The slides were again rinsed in PBS, counter-stained lightlywith Gill's #1 hematoxylin (about 30 seconds), and then rinsed in tapwater. Following dehydration in 2 changes of graded ethanol to 100% for2 minutes each, the sections were cleared in 4 changes of 100% xylenefor 2 minutes each and mounted with Fisher permount.

PCR amplification of ligA in pathogenic serovars. Using a primer pairspecific for ligA, PCR was performed on pathogenic serovars including L.interrogans serovar pomona, type kennewicki, L. kirschneri serovargrippotyphosa, L. interrogans serovar hardjo, type hardjobovis, L.interrogans serovar icterohaemorrhagiae and L. interrogans serovarcanicola. The forward primer was “5′-GGAATTCATGTTAAAGTCACTGCT-3′” (SEQID NO:39) and the reverse was “5′-CCGCTCGAGGTTTTAATAGAGGC-3′” (SEQ IDNO:40). Amplification conditions were as previously described (Chang,Y.-F. et al., Vet. Pathol., 37:68-76 (2000)). PCR products were purifiedusing a gel-purification kit (Qiagen) and digested with BamH1 andHindIII to detect restriction polymorphisms.

Enzyme-linked immunosorbent assay (ELISA). Wells of 96 well polystyreneplates (Falcon 3912 Microtest III, Becton Dickinson, Oxnard, Calif.)were coated overnight at 4 ° C. with 0.15 μg truncated recombinant LigAin 100 μl PBS, washed, blocked with 2% skim milk in PBS (pH 7.2) with0.05% Tween 20 and then incubated with a 1: 100 dilution of horse serumin triplicate wells for 2 hours at 37° C. After washing, peroxidaseconjugated protein G (1:8000) was added (100 μl) to each well andincubated for 2 hours at 37 ° C. Finally, the plates were washed anddeveloped with fresh substrate consisting of 0.07% orthophenylenediamineand 0.05% hydrogen-peroxide in citric acid-phosphate buffer (pH 5.0).After stopping the reaction with 50 μl 3M sulfuric acid, absorbance wasread at 490 nm in an automated plate reader (Biotex, Winooski, Vt.).

Statistical Analysis.

Analysis of variance was used to determine whether there was asignificant difference in the mean OD reading for each of the sera usedin this study. Multiple comparisons using the least significantdifference method were performed to identify which OD mean wassignificantly different from the other. The analysis was performed usingthe Statistix software (Analytical Software, Tallahassee, Fla.).

Nucleotide sequence accession numbers. The GenBank accession number forthe nucleotide sequences of ligA is AF368236.

Results

Identification, sequencing and expression of LigA. Screening of the L.interrogans genomic library with convalescent mare's serum yieldednumerous positive clones, one of which contained an insert of 3,993 bpand expressed a protein that was encoded by an open reading frame of3,675 bp (FIG. 1). The deduced sequence consisted of 1,225 amino acidswith an estimated molecular mass of 129,041 daltons and a pI of 6.35. AnN-terminal signal sequence of 31 amino acids was predicted using theSignal P program (Nielsen, H. et al., Protein Eng., 10:1-6 (1997)).Twelve or more tandem repeats of 90 amino acids were detected in LigA(FIG. 2). Analysis of the sequence using NCBI and BLAST revealedhomology with the immunoglobulin-like domain of E. coli intimin (Genbankaccession number AF252560), the putative invasin of Yersinia pestis(AJ41459) and the cell adhesion domain of Clostridium acetobutylicum(AE007823) (data not shown). LigA tandem repeats that showed homologywith bacterial Ig-like domains (Igl1, CD:pfam02368; Igl2, CD:smart00635) are represented in FIG. 2.

Expression of LigA in E. coli but not in Leptospira lysates. E. colicontaining intact ligA without its signal sequence expressed LigA onlyafter IPTG induction (FIG. 3), but LigA expression was toxic to E. coliresulting in a 50 fold decrease in viability of cells (data not shown),which is similar to OmpL1 of Leptospira (Haake, D. A. et al., Infect.Immun., 67:6572-6582 (1999)). However, the expression of a 90 kDatruncated LigA was not toxic to E. coli cells (data not shown).Immunoblotting of whole cell lysates of L. interrogans serovar pomonatype kennewicki grown at 30 and 37° C. with LigA specific polyclonalrabbit serum did not show any detectable level of LigA (FIG. 4). Incontrast, LipL32 was expressed by cultures grown at both 30 and 37° C.whereas LipL36 was down regulated at 37° C.

LigA expression in vivo in Leptospira-infected hamsters. In order toexamine LigA expression during leptospiral infection,immunohistochemistry was performed on kidneys from normal andleptospiral-infected hamsters. LigA was expressed only inleptospiral-infected hamster kidney (FIG. 5A). High titer rabbitanti-leptospiral serum as well as antiserum to LipL32 reacted withleptospires in experimentally infected kidney (FIGS. 5B and 5C). LipL36,which is not expressed by L. krischneri serovar grippotyphosa ininfected hamster kidney (Barnett, J. K. et al., Infect. Immun.,67:853-861 (1999)), was detected around the proximal convoluted tubulesin L. interrogans serovar pomona infected hamster kidney at a 1:50dilution of antiserum to LipL36 (FIG. 5D). Pre-immune rabbit serum didnot react (FIG. 5E) and no immune serum reacted with normal hamsterkidney (data not shown).

LigA specific antibody in sera of convalescent mares and abortedfetuses. All convalescent sera showed strong reactivity with recombinantLigA by western blot analysis. Negative control horse sera derived fromBorrelia burgdorferi (Chang, Y.-F. et al., Vet. Pathol., 37:68-76(2000)), Human Granuloctyic Ehrlichiosis agent (HGE) infection (Chang,Y.-F. et al., Vet. Parasitol., 78:137-145 (1998)) and naïve horse serawere unreactive (FIG. 6). The utilization of LigA in ELISA also showedstrong reactivity to the convalescent sera (Table 1 below). The mean ODfor the leptospiral positive sera (M1-M8) was significantly differentfrom the negative control (L1-L-5) and from sera obtained from HGE(E1-E2) and B. burgdorferi (N1-N4) infected animals (P<0.05).

TABLE 1 Reactivity in ELISA of rabbit antiserum to recombinant LigA,sera from horses infected with B. burgdorferi (L1-5) or E. Equi (E1 and2), normal horse sera (N1-4) and aborted mare's sera (M1-8) in ELISAwith a 90 kDa truncated LigA (200 ng/well). The ELISA OD values of serafrom aborted mares were significantly higher (P < 0.05) than the valuesfor sera from normal, B. burgdorferi and E. equi infected horses. ELISAOD at serum dilution Serum 1/200 1/400 1/800 Rabbit antiserum to a 901.13 1.02 .58 kDa truncated LigA L1 .05 .03 .01 L2 .1 .04 .02 L3 .03 .02.02 L4 .05 .02 .03 L5 .02 .01 .01 E1 .05 .03 .05 E2 .08 .05 .04 N1 .01.01 0.0 N2 .01 .0 .0 N3 .02 .01 .01 N4 .03 .03 .01 M1 .39 .34 .19 M2 .38.35 .18 M3 .45 .31 .2 M4 .6 .56 .27 M5 .28 .2 .13 M6 .47 .56 .4 M7 .73.55 .4 M8 .56 .5 .42

Detection of ligA in other serovars by PCR. PCR amplification revealedthe presence of ligA in genomic DNA of the pathogenic serovars hardjo,grippotyphosa, icterohaemorrhagiae and canicola (FIG. 7A). Restrictionanalysis with BamHI revealed no differences in fragment patterns.However, HindIII digests revealed that ligA was more highly conserved inL. interrogans serovar pomona and L. kirchneri serovar grippotyphosathan in other serovars (FIG. 7B).

Discussion

LigA is mostly hydrophilic with some hydrophobic regions located atresidues 4-24, 306-326, 402-422, 490-510 and 1034-1054 (FIG. 1) andconsists of beta sheets with a few alpha helical regions. AnAla-Lys-Glu-Leu-Thr (SEQ ID NO:41) peptide repeat occurs at positions416, 505, 594 and 867 corresponding to alpha helices. LigA contains 12or more tandem repeats of a 90 amino acid sequence (FIG. 2). Analysis ofthe nucleotide sequences using NCBI and BLAST revealed no homology otherthan that between the repeat region of LigA and the immunoglobulin-likedomain of intimin binding protein (int) of E. coli (Hamburger, Z. A. etal., Science, 286:291-295 (1999); Kelly, G. et al., Nat. Struct. Biol.,6:313-318 (1999); Luo, Y. et al., Nature, 405:1073-1077 (2000)), theinvasin of Yersinia pestis (Isberg, R. R. et al., Cell, 50:769-778(1999); Jerse, A. E. and J. B. Kaper, Infect. Immun., 59:4302-4309(1991)) and a cell binding domain of Clostridium acetobutylicum(Nolling, J. et al., J. Bacteriol., 183:4823-4838 (2001)).

Although sera from recently aborted mares reacted strongly with the 90kDa truncated LigA, the protein was not detectable by immunoblot inLeptospira lysates cultured at 30° and 37° C. In contrast, LipL32 isexpressed at both 30° and 37° C. while LipL36 expression is growth-phasedependant (Haake, D. A. et al., Infect. Immun., 68:2276-2285 (2000);Nally, J. E. et al., Infect. Immun., 69:400-404 (2001)). This indicatesthat LigA is not expressed or thermo-regulated under in vitro cultureconditions.

However, immunohistochemistry using rabbit antiserum specific for a 90kDa truncated LigA revealed expression of LigA in kidneys of infectedbut not uninfected hamsters. A commercially available high titeranti-leptopspiral antiserum showed strong reactivity to the leptospiralorganisms in infected hamster kidney. Expression of LipL32 was detectedboth in vitro (culture) and in vivo (leptospiral-infected hamsterkidney) whereas LipL36 expression has been reported only in vitro(Barnett, J. K. et al., Infect. Immun., 67:853-861 (1999)). The in vivoexpression of LipL32 has also been confirmed. However, the reactivity ofLipL36 rabbit polyclonal antibody with infected hamster kidney at a 1:50dilution was noted. In contrast, Barnett et al. failed to detectexpression of LipL36 in L. kirschneri serovar grippotyphosa infectedhamster kidney. These positive controls confirm that LigA is expressedonly in vivo.

A 90 kDa protein of Leptospira has been previously shown to cross-reactwith polyclonal antiserum to an equine corneal protein (Lucchesi, P. M.and A. E. Parma, Vet. Immunol. Immunopathol., 71:173-179 (1999)).Immunohistochemistry, immunoprecipitation and Western blot analysisrevealed no reactivity of LigA specific antiserum with equine cornea,iris, vitreous or lens (data not shown). Thus, LigA does not appear toshare antigenic epitopes with equine ocular components and so it isclearly not the reactive protein (Lucchesi, P. M. and A. E. Parma, Vet.Immunol. Immunopathol., 71:173-179 (1999)).

PCR amplification of ligA from genomic DNA of pathogenic serovars suchas hardjo, icterohaemorrhagiae, grippotyphosa, and canicola has shownthat a similar sequence is widely distributed among the serovars of L.interrogans. However, restriction analysis with HindIII showed that theligA sequence had greater similarity to that of serovars pomona andgrippotyphosa than to serovars canicola and icterohaemorrhagiae.Interestingly, L. interrogans serovar pomona and L. kirchneri serovargrippotyphosa are the serovars most frequently responsible for diseasein the horse.

The expression of outer membrane proteins of Leptospira such as LipL32,LipL41, OmpL1 and LipL36 has been demonstrated in cultured organisms(Haake, D. A. et al., J. Bacteriol., 175:4225-4234 (1993); Haake, D. A.et al., Infect. Immun., 68:2276-2285 (2000); Haake, D. A. et al.,Infect. Immun., 66:1579-1587 (1998); Haake, D. A. et al., Infect.Immun., 67:6572-6582 (1999)). Except for LipL36, these outer membraneproteins are expressed in infected hamsters. Interestingly, this is thefirst leptospiral protein that is not detectable in vitro (30 or 37° C.)but is expressed in kidneys of infected hamsters.

Example II Identification of LigB

ligB was obtained using the same procedures as were used to obtain ligA.To summarize, a genomic library of L. interrogans serovar Pomona typeKennewicki was constructed as previously described and was screened withconvalescent sera from leptospiral infected horses and mares thataborted due to leptospirosis (Palaniappan, R. U. M. et al., Infect.Immun., accepted (2002)). Several positive clones were identified andone of the recombinant clones contained an open reading frame (ORF) of4200 bp (FIG. 8). The deduced sequence contained 1,420 amino acids withan estimated molecular weight of 140 kDa (SEQ ID NO:4). An N-terminalsignal sequence of 31 aa was predicted using the Signal P program(Nielsen, H. et al., Protein Eng., 10:1-6 (1997)). Three possible startcodons for this protein were identified and upstream of the start codonof ligB is a potential ribosome-binding site (FIG. 8). NCBI Blast searchrevealed homology with the conserved bacterial immunoglobulin-likedomain (Pfam Big 2) of intimins from E. coli (AF319597, AF301015,AF116899) and cell adhesion domain from C. acetobutylicum(NC_(—)003030). Nucleotide sequence accession numbers. The GenBankaccession number for the nucleotide sequences of ligB is AF534640.

Example III Comparison of LigA and LigB

LigB has complete homology with LigA in the N-terminal sequences (up to630 amino acids) but is variable in the carboxyl terminal. Thestructural analysis reveals that LigA and LigB are present on thesurface of Leptospira. Interestingly, LigB contains twelve 90 amino acidsequence repeats whereas LigA consists of thirteen repeats. In addition,LigB contains an agglutinin-like domain (lectin type) from residues1054-1160, and a possible tyrosine kinase phosphorylation site fromresidues 1150-1158 (KEALDLSNY; SEQ ID NO:42). The comparison of intiminbinding domain of translocated intimin receptor (Tir) (272-304 residues)to LigB using Cn3D, NCBI revealed 25% homology to LigB (1353-1378residues).

LigA and LigB are similar to intimin with a homology of 24%. Intimin hasa Lys motif, two Ig-like domains, Dl and D2 (residues 658-751 &752-841), and a C-type lectin-like domain D3 (residues 752-841).Similarly, Invasin has four Ig-like domains (D1-D4) and a C-typelectin-like domain (D5) (Bjorkman et al., 1999) whereas LigA and LigBconsists of thirteen (D1-13) and twelve repeats (D1-D12) of 90 aminoacids motif respectively which have homology to the bacterial domainswith Ig-like fold (pfam Big2). Additionally, LigB contained a C-typelectin-like domain, D13 (residues 1014-1165) (FIG. 9). Recently, BipAfrom Bordetella bronchiseptica has been reported to contain 8 tandemrepeats of 90 amino acids with a lectin-like carboxyl terminal(Stockbauer, K. E., et al., Mol. Microbiol., 39:67-78 (2001)). Theintimin shares 24% homology with LigA and LigB respectively. Twelverepeats of LigB and its homology with bacterial Ig-like domain aredepicted in FIG. 10.

Interestingly, LigB is identical in the N-terminal sequences (up to 630amino acids) with LigA but varies in the carboxyl terminal (FIG. 11).The first 5 tandem repeats (residues 52-133, 137-222, 226-308, 312-398,402-487 and 491-576) at the N-terminal regions of LigA and LigB are thesame. Furthermore, a C-type lectin-like domain, especially the aminoacids KEALDLSNY (SEQ ID NO:42; residues 1150-1158) contains tyrosinekinase phosphorylation sites according to the Scanprosite program(SWISSPROT). The alteration in the number of tandem repeats andvariation in the carboxyl terminal of LigA and LigB may modify theirantigenic determinants to evade the host immune response (Jones, C. J.,West Indian Med. J. 23:65-68 (1974); Duncan, L. R. et al., Mol. Biochem.Parasitol., 48:11-16 (1991)).

The induction of attachment-effacing lesions by bacteria involves athree-stage model including initial attachment of bacteria to hostcells, signal transduction and phosphorylation of host cells, andfinally, intimate attachment of bacteria to the host cell membrane.Yersinia and E. coli mediate internalization into host cells usingsurface proteins such as invasin and intimin (Isberg, R. R. et al.,Cell, 50:769-778 (1987); Jerse, A. E. et al., Proc. Natl. Acad. Sci.U.S.A., 87:7839-7843 (1990)). Invasin mediates entry by binding tointegrins that activate reorganization of the cytoskeleton (Tran VanNhieu, G. and Isberg, R. R., Embo J., 12:1887-1895 (1993)). InEnteropathogenic E. coli (EPEC), intimin binds to Tir (translocatedintimin receptor) produced by this bacterium, which eventually inducescytoskeletal rearrangements (Kenny, B. et al., Cell, 91:511-520 (1997)).Although the role of tyrosine phosphorylation in pedestal formation isunclear, Tir is tyrosin phosphorylated in EPEC but not inEnterohemorrhagic E, coli (EHEC) (DeVinney, R. et al., Cell Mol. LifeSci., 55:961-976 (1999)). It has been indicated that synthesis andsecretion may be differentially regulated in these pathogens (DeVinney,R. et al., Cell Mol. Life Sci., 55:961-976 (1999)). These proteins areencoded by the genes in pathogenicity island, also called as locus ofenterocyte effacement (LEE) (McDaniel, T. K. et al., Proc. Natl. Acad.Sci. U.S.A., 92:1664-1668 (1995)). LigB also contained a potentialtyrosine kinase phosphorylation site but the localization of the gene inthe pathogenicity island has not yet been unraveled. NCBI, Cn3D analysisrevealed 25% homology to intimin binding region of translocated intiminreceptor at the carboxyl terminal (residues 272-304) especially 32 aminoacids with C-type lectin like domain of LigB. Since LigB contains anagglutinin-(lectin type) like domain with a possible tyrosinephosphorylation site at the carboxyl terminal and Tir like receptor, itseems that LigB may trigger cellular signaling events in the host cell.

The hydrophobicity of the deduced amino acid sequence and theirpotential membrane-spanning region was analyzed. Lig A and B are mostlyhydrophilic with some hydrophobic regions and they consist of betasheets with a few alpha helical regions. The predicted transmembraneregion of LigB is from 300-319 residues (IIGSVKLIVTPAALVSI) (SEQ IDNO:43). Cysteine reportedly plays an important role in integrin bindingand protein folding (Leong, J. M. et al., J. Biol. Chem.,268:20524-20532 (1993)); Frankel, G. et al., J. Biol. Chem.,271:20359-20364 (1996)). Invasin (Cys906 and Cys982) and intimin (Cys860and Cys937) contain 2 cysteines and mutants lacking cysteine fail tointeract with eukaryotic cells. Analysis of amino acids in the carboxylterminal of Lig A and B revealed two molecules of cysteine in LigAwhereas LigB contains eight cysteines. The numbers of serine andthreonine residues in LigB are 224 and 147 whereas LigA has 179 and 142.Regardless, they are the most dominant amino acids in both of theseproteins. Similar to invasin and intimin, LigB lacks an Arg-Gly-Aspsequence (RGD), which is critical for the interaction of fibronectinFn-III 10 with integrins (Hynes, R. O., Cell, 69:11-25 (1992)). However,Asp911 of invasin is critical for integrin binding (Hamburger, Z. A. etal., Science, 286:291-295 (1999)). A WIGL (trp-ile-glu-leu; SEQ IDNO:44) sequence, characteristic for calcium-coordinating residues thatare critical for carbohydrate recognition, is not present in intimin,invasin (Hamburger, Z. A. et al., Science, 286:291-295 (1999)) and isalso missing from LigB.

Example IV Expression of Leptospiral Immunoglobulin-like Protein fromLeptospira interrogans and Evaluation of its Diagnostic Potential inKinetic Enzyme Linked Immunosorbent Assay

Summary

The search for vaccine/diagnostic antigens against leptospirosis led tothe identification of LigA (Palaniappan et al., Infect. Immun.,70:5924-5930, 2002). Similar to ligA, the ligB gene was obtained byscreening a genomic library of L. interrogans with convalescent sera.The ligB gene contains an open reading frame of 5,667 bases that encodes1,889 amino acids. LigB has complete homology with LigA at the aminoterminal region, but is variable at the carboxyl terminal. LigB containstwelve, 90 amino acid sequence repeats of an immunoglobulin-like foldand an agglutinin-like domain (lectin type). Structural analysisrevealed that LigA and LigB are surface proteins. Lig genes were presentin most of the pathogenic serovars of Leptospira, but not in thenon-pathogenic L. biflexa. LigA and LigB expression were not detectableat the translational level, but were detectable at the transcriptionallevel in in vitro grown leptospires. The conserved region and variableregions of LigA and LigB (Con, VarA and VarB) were cloned and expressedas GST fusion proteins. Kinetics-ELISA (KELA) was performed with GSTfusion proteins of Con, VarA and VarB. Ninety-four canine sera positivefor leptospirosis by MAT were evaluated in KELA with Con, VarA and VarB.Out of ninety-four, fifty-six MAT positive canine sera were found to bereactive in KELA. The conserved region of LigA and LigB showed strongerreactivity in KELA than variable regions of LigA and LigB. Canine serawith a MAT titer of >1,600 showed reactivity of 76% to Con, 41% to VarAand 35% to VarB respectively in KELA, suggesting the suitability ofthese antigens for the serological diagnosis of leptospirosis.

Leptospirosis is caused by spirochetes belonging to the genusLeptospira. considered the most widespread zoonotic disease in the world(World Health Organization, 1999). Leptospirosis affects both humans andanimals (Vinetz, Curr. Opin. Infect. Dis., 14:527-38 (2001)). Infectionis mainly contracted by exposure to water, food or soil contaminatedwith the urine from infected animals (Levett, Clin. Microbiol. Rev.,14:296-326 (2001)). Potential carriers of Leptospira include rats,cattle, dogs, horses, and pigs (Goldstein and Charon, 1990).Leptospirosis in dogs is recognized as a risk factor for humanleptospirosis (Douglin et al., 1997). Increased rainfall is associatedwith a rise in the prevalence of leptospirosis in dogs (Ward, Prev. Vet.Med., 56:215-26 (2202)). Infection can lead to pulmonary hemorrhage,renal, hepatic failure and/or multi-organ failure and even death(Levett, Clin. Microbiol. Rev., 14:296-326 (2001)). An infected dog canalso act as an asymptomatic carrier and shed infectious organisms in theurine for its entire lifetime (Murray, Vet. Rec., 127:543-7 (1990)).Approximately 250 serovars have been identified. The availableleptospiral vaccines, however, elicit only short-term immunity and donot provide cross protection against different serovars.

Diagnosis of leptospirosis is complicated by the high degree ofcross-reaction between different serovars of Leptospira. Furthermore,the non-pathogenic L. biflexa serovar Patoc, considered an environmentalcontaminant, provides a cross-reactive pattern to rabbit sera frompathogenic serovars of Leptospira (Matsuo et al., Microbiol. Immunol.,44:887-90 (2000); Myers, J. Clin. MicroBiol., 3:548-55 (1976); Myers andColtorti, J. Clin. Microbiol., 8:580-90 (1978)). Currently availablediagnostic techniques include the microscopic agglutination test (MAT),which is laborious and not widely available. In addition, ELISA methodshave been developed with number of modifications (da Silva et al., Am.J. Trop. Med. Hyg., 56:650-5 (1997); Gussenhoven et al., J. Clin.Microbiol., 35:92-7 (1997); Hartman et al., Vet. Immunol. Immunopathol.,7:43-51 (1984); Hartman et al., Vet. Immunol. Immunopathol., 7:33-42(1984); Levett, Clin. Mictobiol. Rev., 14:296-326 (2001); Petchclai etal., Am. J. Trop. Med. Hyg., 45:672-5 (1991); Ribeiro et al., J. Trop.Med. Hyg., 98:452-6 (1995)), but most of them depend on the whole cellproteins of Leptospira. Recombinant antigens such as LipL32, flagellinand heat shock protein of Leptospira have also been recently developedfor diagnosis (Flannery et al., J. Clin. Microbiol, 39:3303-10 (2001);Park et al., DNA Cell Biol., 18:903-10 (1999)), but the specificity andsensitivity of these antigens in vaccinated animals have not beendetermined. The major drawback with the MAT and ELISA procedures is thatthey cannot differentiate between infected and vaccinated animals.Identification of leptospiral antigens expressed only during infectioncould be used for the development of new diagnostic reagents thatdifferentiate between vaccinated and infected animals.

In order to identify antigens that are expressed during leptospiralinfection, a genomic library of L. interrogans was screened with serafrom infected animals. Several positive clones were obtained. One cloneencoded a gene for a leptospiral immunoglobulin like proteins, referredto as LigA, that is only expressed in vivo (Palaniappan et al., Infect.Immun., 70:5924-30 (2002)).

The present application discloses the identification of anotherleptospiral immunoglobulin-like protein, named LigB. LigB is identicalto LigA at the amino terminus, but is variable at the carboxyl terminus.Truncated forms of the conserved region (Con) and variable regions ofLigA (VarA) and LigB (VarB) were expressed as GST fusion proteins in E.coli. These recombinant antigens were used in a computer controlledkinetics-based enzyme linked immunosorbent assay (KELA) and wereevaluated for their diagnostic potential in vaccinated and MAT positivecanine sera. Data disclosed herein indicate that these recombinantantigens can serve as diagnostic reagents for the detection ofleptospiral infection.

Materials and Methods

Sera. Convalescent sera obtained from mares that had recently aborteddue to leptospiral infection were used to a screen genomic library of L.interrogans. These sera have high titers for L. interrogans serovarPomona as determined by the microscopic agglutination test.

A total of 94 canine sera positive for leptospirosis by MAT (MATpositive canine sera) were collected from 1999 to 2002 from the New YorkState Animal Health Diagnostic Laboratory at Cornell University, Ithaca,N.Y.

Vaccinated sera were obtained from eight week old puppies that had beenvaccinated with commercially available vaccines, such asGrippotyphosa/Pomona (G/P), Canicola/Icteroheamorragiae (C/IC) andGrippotyphosa, Pomona, Canicola and Icterohaemorragiae (GPIC) followedby booster injection three weeks later. Sera were collected beforevaccination and on the 5^(th) and 9^(th) week after vaccination.

Control sera were obtained from dogs naturally infected with Leishmaniadonovoni, Borrelia burgdorferi or Trypanosoma cruzi and stored at NewYork State Diagnostic laboratory at Cornell University, Ithaca, N.Y.

Sera were also collected from specific pathogen free beagles (SPF) andalso lyme-vaccinated dogs (Chang et al., Infect. Immun., 63:3543-3549(1995); Chang et al., Am. J. Vet. Res., 62:1104-1112 (2001)).

Bacterial strains and culture conditions. L. interrogans serovar Pomonatype kennewicki was isolated from an equine abortion (Wen et al.,Nature, 422:888-893 (2003)). Leptospires were maintained on PLM-5 medium(Intergen, N.J.), at 30° C. To isolate low passage cultures ofleptospires, experimentally infected hamster tissues were homogenizedand inoculated into PLM-5 medium. High passage cultures were prepared byrepeated passage (<15 times) of leptospires in PLM-5 medium. Growth wasmonitored by dark field microscopy.

DNA sequencing and analysis. The positive clones containing ligB gene(derived from screening a genomic library, as previously described) weresubjected to DNA sequencing (Palaniappan et al., Infect. Immun.,70:5924-5930 (2002)). DNA sequencing was done using an ABI model 377automated nucleic acid sequencer at the Bioresource Center, CornellUniversity, Ithaca, N.Y. Homology searches were performed with NCBI,Blast (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997)).

Construction of GST fusion proteins of Lig A and LigB. LigA and LigBwere truncated into conserved (Con, the N-terminal 599 amino acidswithout the signal sequences) and variable regions (VarA and VarB, theC-terminal 595 and 788 amino acids of LigA and LigB, respectively). Theregions were amplified using PCR with the following primers ligConF.5′-]“TCCCCCGGGGCTGGCAAAAGA,” (SEQ ID NO:47) ligConR.5′-“CCCTCGAGAATATCCGTATTAGA,” (SEQ ID NO:48) VariAF,5′-“CCCCCGGGCTTACCGTTCC,” (SEQ ID NO:49) VariAR,5′-“CCCTCGAGTGGCTCCGTTTTAAT,” (SEQ ID NO:36) VariBF,5′-“TCCCCCGGGGCTGAAATTACAAAT,” (SEQ ID NO:50) VariBR, 5′-“CCGCTCGAGTTGGTTTCCTTTTACGTT” (SEQ ID NO:51). The underline nucleotides indicatethe restriction site. PCR was performed using 0.5 units accuprime Taqpolymerase (Invitrogen, CA). Other reagents were added as outlined bythe manufacturer's instructions (Invitrogen, CA). The reaction mixturewas subjected to 35 cycles after an initial denaturation at 94° C. for 5minutes. Each cycle consisted of 94° C. for 1 minute, 50° C. for 2minutes and 72° C. for 5 minutes.

PCR products were subcloned using a TOPO TA cloning kit (Invitrogen,Carlsbad, Calif.) into pGEX4T-2 plasmids (Amersham Pharmacia). Therecombinant plasmids (pLigCon, pLigVarA, pLigVarB) were then introducedinto E. coli BL21 (DE3). The resulting transformants were grown at 37°C. overnight on LB agar plates containing 50 μg/mL ampicillin. Theexpression of proteins was induced with 1mM IPTG.

Purification of GST fusion proteins. IPTG induced E. coli BL21 (DE3)containing the recombinant plasmids was harvested by centrifugation at5000 rpm. The cell pellets were washed and suspended in PBS followed bypassing through a French pressure cell (American Instrument, SilverSpring, Md.). The lysates were then centrifuged to remove the celldebris, and the supernatants were subjected to affinity chromatographyusing glutathione-Sepharose 4B columns. (Amersham Pharmacia Biotech,Piscataway, N.J.). The GST fusion proteins were eluted according to themanufacturer's instructions (Amersham Pharmacia Biotech, Piscataway,N.J.).

Generation of Polyclonal Antibodies. Adult New Zealand white rabbitswere immunized intramuscularly with 100 μg of GST fusion proteins and anequal amount of Freund's incomplete adjuvant. Rabbits were boostedsubcutaneously with the same dosage on the 19^(th) and 35^(th) day. Onday 45, the rabbits were bled, and the sera were collected for analysis.

SDS-PAGE and Immunoblot analysis. The recombinant proteins weresubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresisfollowed by immunoblot as previously described (Chang et al., Infect.Immun., 63:3343-9 (1995); Chang et al., Am. J. Vet. Res., 62:1104-12(2001); Chang et al., DNA Cell Biol., 12:351-62 (1993)).

RT-PCR. RNA was isolated from log phase cultures of leptospires using anRNA mini kit (Qiagen Inc.), treated with RNAase free DNAase, andsubjected to one step RT-PCR with gene specific primers (variable regionof ligA and B). The following primers were used for RT-PCR: VariAF,5′-GAAAATCGCATCAGTAGAAAAC (SEQ ID NO:52); VariAR, 5′-CCCTCGAGTGGCTCCGTTTTAAT (SEQ ID NO:36), VariBF, 5′-TAAACAAAACGGACACGATAGC (SEQ ID NO:53);VariBR, 5′-CCGCTCGAGTTGGTTTCCTTTTACGTT (SEQ ID NO:51). The reactionswere carried out according to the manufacturer's instructions (Qiagen).A reaction containing all the reagents except for reverse transcriptasewas used as a negative control. Genomic DNA was used as a positivecontrol.

Southern blot analysis. Genomic DNA isolated from leptospiral strainswas digested with EcoRI and subjected to gel electrophoresis. DNA wastransferred to Hybond N+ nitrocellulose membranes (Amersham Pharmacia).The membranes were processed as outlined in the manufacturer'sinstructions for ECL direct nuclei labeling and detection system(Amersham Pharmacia, NJ). The conserved region of the lig gene was usedas the probe for the Southern blot analysis.

Optimization of antigen concentration. Based on MAT titer, canine serawere categorized into high (MAT titer of 12,800 to Pomona, 6,400 toGrippotyphosa), low (MAT titer of 6,400 to Pomona, 3,200 toGrippotyphosa) and negative (SPF, specific pathogen free serum). Acheckerboard titration of recombinant antigens (Con, VarA and VarB),primary antibodies (negative, medium and high) and secondary conjugate(anti-dog conjugated with horseradish peroxidase) was performed todetermine the optimum conditions.

Kinetic ELISA (KELA). The optimized concentrations of recombinantantigens were diluted in 0.1 M bicarbonate buffer and added to a 96 wellmicrotiter plate (Nunc, Denmark). The plates were rocked for 1 hour andthen incubated overnight at 4° C. The plates were washed three timeswith 0.1M PBS containing 0.05% Tween 20 (PBST). Canine sera (primaryantibody) in PBST were diluted to 1:200. 100 μl of diluted serum wasadded to each well and the plates were incubated for 1 hour at 37° C. ina humid chamber. The plates were washed three times with PBST, and thenincubated with 100 μl of a 1:4000 dilution of goat anti-dog IgGconjugated to horseradish peroxidase (Cappel, Durham, N.C.) for 30minutes at room temperature. The plates were washed again three timeswith PBST, and 100 μl of TMB (Kirkegaard, Md.) was added to each well.Each plate was read three times in a microplate spectrophotometer(Bio-Tek EL-312, Winoski, Vt.) at 650 nm OD with an interval of1-minute. The results were calculated by the KELA computer program(Diagnostic Laboraotry, College of Veterinary Medicine, CornellUniversity) and expressed as the slope of the reaction between enzymeand substrate to the amount of antibody bound (Chang et al., Infect.Immun., 63:3543-3549 (1995); Chang et al., DNA Cell Biol., 12:351-362(1993)).

Statistical analysis. The significance of differences between therecombinant proteins in relation to KELA units was evaluated using theanalysis of variance statistical method. The analysis was performed inSTATISTIX (Analytical software, Tallahassee, Fla.). The least squaredifference post-hoc test was used to determine mean KELA value and thesignificant difference of the recombinant proteins. The correlationbetween MAT and KELA for the six serovars of Leptospira was evaluatedusing the Pearsons Correlation in STATISTIX. This correlation wasassessed for each recombinant protein separately. Descriptive statisticswere performed to determine the cut off value for each protein inrelation to KELA.

Nucleotide Sequence Accession Number. The Genbank accession number forthe nucleotide sequence of ligB is AF534640.

Results

Identification, sequencing and expression of LigB. A leptospiral genomiclibrary was constructed as previously described. The library wasscreened with convalescent sera obtained from leptospiral-infected maresthat had aborted (Palaniappan et al., Infect. Immun., 70:5924-5930(2002)). Several positive clones were identified, one of which containedan open reading frame (ORF) of 5,667 bp. The deduced protein sequencecontained 1,889 amino acids and had an estimated molecular weight of 206kDa. An N-terminal signal sequence of 31 amino acids was predicted usingthe signal P program (Nielsen et al., Protein Eng., 10:1-6 (1997),Nielsen et al., J. Am. Vet. Med. Assoc., 199:351-352 (1991)). Threepossible start codons for this protein were identified and upstream ofthe start codon of ligB is a potential ribosome-binding site. Therecently released genomic sequences of L. icterohaemorrahgiae serovarlai contained LigB but not LigA (Genbank number AA065920), which shows98% homology with LigB of L. interrogans serovar Pomona (Ren et al.,Nature, 422:888-893 (2003). NCBI Blast search revealed homology with theconserved bacterial immunoglobin-like domain (Pfam Big 2) of intiminsfrom E. coli (AF319597, AF301015, AF 116899) and cell adhesion domainfrom Clostridium acetobutylicum (NC-003030).

Primary structure of LigB and comparison with LigA. LigB consists oftwelve repeats (D1-D12) of a 90 amino acid motif, which has homology tothe bacterial domains with Ig-like fold (pfam Big2) (FIG. 13A). It hasbeen reported that LigA contains twelve repeats of a 90 amino acidmotif, but according to pfam, LigA actually has thirteen repeats of the90 amino acid motif. Additionally, LigB contains a C-type lectin-likedomain, D13 (residues 1014-1165).

The amino terminal sequence (the first 630 amino acids) of LigB isidentical to LigA, but the carboxyl terminus varies (FIG. 13B). Thefirst 5 tandem repeats (residues 52-133, 137-222, 226-308, 312-398,402-487 and 491-576) at the N-terminal regions of LigA and LigB areidentical. Furthermore, a C-type lectin-like domain, especially theamino acids KEALDLSNY (residues 1150-1158), contains tyrosine kinasephosphorylation sites according to the Scanprosite program (SWISSPROT).The numbers of serine and threonine residues in LigB are 224 and 147,whereas LigA has 179 and 142, respectively. Regardless, serine andthreonine are the most dominant amino acids in both of these proteins.

The hydrophobicity of the deduced amino acid sequence and the potentialmembrane-spanning region of LigA and B was analyzed. These two proteinsare largely hydrophilic with some hydrophobic patches, and they consistof beta sheets with a few alpha helical regions. The predictedtransmembrane region of LigB spans residues 300-319 (IIGSVKLIVTPAALVSI)(SEQ ID NO:43).

lig genes are widely spread, but only in pathogenic serovars. In orderto determine the presence of lig genes in different serovars ofLeptospira, EcoRI digested genomic DNA from different serovars ofLeptospira was transferred to nitrocellulose membranes, and probed witha non-radioactively labeled oligonucleotide from the conserved regionsof LigA and LigB. Non-pathogenic L. biflexa serovar Patoc did notcontain lig genes, but the other pathogenic serovars contained copies oflig genes (FIG. 14).

Expression and purification of LigA and B. In order to over express LigAand B, the truncated forms of the conserved and variable regions of LigAand B were cloned and expressed as GST fusion proteins. The expressedrecombinant proteins of the conserved and variable regions of LigA andLigB had molecular weights of 92, 93, and 120 kDa, respectively. GSTfusion proteins were purified using affinity column chromatography andthrombin cleaved proteins migrated as 62, 63, and 82 kDa, respectively(FIG. 15A, B and C).

Lack of LigA and LigB expression at the translation level inleptospires. To examine LigA and B expression in leptospires,immunoblots of whole cell proteins of low and high passage cultures ofleptospires were probed with polyclonal antibodies to Con, VarA andVarB. LigA and LigB expression was not detectable in leptospires grownin vitro (FIG. 16A, B and C). In contrast, E. coli containing therecombinant plasmids showed strong reactivity. The negative control, E.coli without the insert in the vector, showed no reactivity.

Detection of LigA and B at the transcript level in leptospires grown invitro. RT-PCR with RNA of low passage and high passage cultures detectedthe expression of LigA and LigB at the transcript level (FIG. 17).Genomic DNA of leptospires was used as a positive control. The negativecontrol, which lacked reverse transcriptase did not show anyamplification. This indicates that LigA and LigB may be poorly expressedunder in vitro conditions or the proteins are very unstable.

Optimization of recombinant antigen concentrations. LigA and LigB weretruncated into the conserved region of LigA and LigB, variable region ofLigA and variable region of LigB and expressed as GST fusion proteins. Acheckerboard titration technique was used to determine the optimalconcentrations of reagents for ELISA. Based on this, 1 μg of recombinantantigen was chosen and the dilution rate of primary and secondaryconjugated antibodies were assessed as 1:200 and 1:4,000, respectively.Since these recombinant antigens were expressed as GST fusion proteins,GST was used as control and the reactivity rate of GST was subtractedfor the analysis of samples.

Determination of cut off value: A total of 20 sera collected fromunvaccinated/healthy dogs were analyzed in KELA with GST, Con, VarA andVarB. The KELA value for the reactivity of unvaccinated sera to therecombinant antigens was obtained by subtracting the reactivity of GST.The cut off value was determined from the unvaccinated dogs usingdescriptive statistical analysis (Table 1). All the sera showed negativeKELA value in KELA with the recombinant antigens except two sera. Themaximum KELA unit of sera from unvaccinated/healthy dogs was consideredas the cut off value. The cut off KELA value of Con, VarA and VarB were7, 42, and 42, respectively.

TABLE 1 Descriptive statistics of KELA value (KELA) to the recombinantantigens in unvaccinated/healthy dogs Con VarA VarB 1 Lower limit 95% CI0 −1.8578 0 2 Upper limit 95% CI 2.0188 10.328 5.525 3 Mean 0.82354.2353 2.5294 4 Standard deviation 2.3247 11.8531 8.5855 5 Maximum 7 4242

Lack of antibodies in the vaccinated sera to recombinant antigens ofLigA and LigB. A serial bleed from dogs vaccinated with commerciallyavailable vaccines showed MAT titer of less than 400 (Table 2).

TABLE 2 MAT titer value for the sera from vaccinated dogs MAT titer toPomona for vaccinated MAT titer to Grippotyphosa for vaccinated serasera Dogs 5.5 wks 7 wks 10 wks 5.5 wks 7 wks 10 wks G/P — — — — — — C/IC— — — — — — GPIC — — — — — — G/P 200 200 — — — — GPIC — — — — — — G/P100 400 — — — 100 C/IC — — — — — — G/P represents dogs vaccinated withGrippotyphosa and Pomona vaccine; GPIC denotes dogs vaccinated withPomona, Grippotyphosa, Icterohaemorrahagiae and Canicola; C/ICrepresents dogs vaccinated with Canicola and Icterohaemorrahagiae (C/IC)

Analysis of these vaccinated sera showed no reactivity to recombinantantigens Con, VarA and VarB in KELA but showed reactivity to the wholecell proteins of Leptospira interrogans in the western blot analysis(FIG. 18) and in ELISA (our unpublished data). In the western blotanalysis with whole cell lysates, most of the vaccinated sera from dogsshowed reactivity with leptospiral antigens. For example, Grippo/Pomonacombined vaccinated sera reacted with whole cell antigens at 66, 50, and42 kDa, whereas naturally infected sera from dogs showed reactivity withleptospires antigens at 66, 42, 33, 32, 27 and 21 kDa (FIG. 18). Thedescriptive statistical analysis of KELA with sera from the vaccinateddogs was below the cut off value (Table 3).

TABLE 3 Descriptive statistics of KELA value (KELA) to the recombinantantigens in vaccinated dogs Con VarA VarB 1 Lower limit 95% CI 0.0809 00.1102 2 Upper limit 95% CI 1.5381 2.077 1.5088 3 Mean 0.8095 0.80950.8095 4 Standard deviation 1.6006 2.786 2.2441 5 Maximum 6 12 12

Reactivity of MAT positive canine sera to recombinant antigens in KELA.The diagnostic potential of recombinant antigens of LigA and LigB todetect leptospiral-infected dogs was assessed in KELA using MAT positivecanine sera. A total of ninety-four MAT positive canine sera werecategorized based on the MAT titer and the reactivity to KELA withrecombinant antigens Con, VarA and VarB was established based on the cutoff value. FIG. 19A, B and C represent the reactivity of MAT positivecanine sera with recombinant proteins Con, VarA and VarB. Therecombinant antigen Con, VarA and VarB in KELA showed reactivity of 76%,41% and 35%, respectively, to canine sera with MAT titer of more than1,600, but the overall sensitivity of recombinant antigens Con, VarA andVarB to MAT-positive canine sera was 58%, 30% and 17%, respectively(Table 4).

TABLE 4 Comparison of efficiency of recombinant antigens in KELA to MATpositive canine sera. Reactivity of recombinant antigens in KELA MATtiter Con VarA VarB 12,800 88% 73% 58% 6,400 81% 27% 22% 3,200 67% 42% 8% 1,600 67% 22% 11% 800 28%  7% 0   400 15%  8% 0   Overall 58% 30%17% The sensitivity of recombinant antigens in KELA was determined fromMAT positive canine sera, which did not have a case history forvaccination. Based on the cut off value the percentage of reactivity ofrecombinant antigens to MAT titer value is represented.

The low titer (below 1,600) MAT-positive canine sera did not showreactivity to the recombinant antigens. Comparing the reactivity ofthese recombinant antigens with MAT positive canine sera, there aresignificant differences between Con, VarA and VarB in the KELA units.The mean KELA units for Con and VarA are not significantly differentfrom each other. Post-hoc tests showed that VarB had significantly lowerKELA units (19.4) in comparison to Con (53.6) and VarA (41.43). Dogsinfected with leishmaniosis, trypanosomosis and borreliosis did not showreactivity to the recombinant antigens suggesting that there is no crossreactivity of these antigens with these agents.

Lack of Correlation between MAT positive and KELA. The correlationbetween MAT and KELA with recombinant antigens of LigA and LigB wasstudied but it showed poor correlation (Table 5).

TABLE 5 Correlation between MAT and KELA units in the clinical samples.Recombinant proteins Bratislava Canicola Grippotyphosa HardjoIcterohaemorrahgiae Pomona Con 0.0107 −0.2491 0.281 0 0.525 0.2454 VarA0.271 −0.2467 −0.0348 0 0.2021 0.3273 VarB 0.2863 −0.1308 −0.1741 00.1129 0.0125 There was poor correlation between MAT and KELA for sixserovars of Leptospira using pearson correlation in statistixDiscussion

On first exposure to pathogenic bacteria, a host's immune systemgenerates antibodies directed against cell surface or membrane antigens.Since the antibodies against cells surface antigens may abrogatecolonization, recombinant antigens from cell surface or membraneproteins may serve as candidates for the development of novel vaccinesand improved diagnostic tests. In this study, LigB was identified byimmuno-screening of a genomic library of L. interrogans serovar Pomona.Its expression in in vitro grown leptospires was studied. The conservedand variable regions of LigA and LigB were expressed as GST fusionproteins, and a KELA test was developed for the detection of leptospiralinfection.

Previously, the lack of expression of LigA in vitro using high passagecultures of leptospires was demonstrated (Palaniappan et al., Infect.Immun., 70:5924-5930 (2002)). Similarly, LigB expression in vitro is notdetectable in high and low passage cultures of leptospires. However,both LigA and B are detectable at the transcript level. The lack ofdetection of LigA and LigB in in vitro grown leptospires suggests thatthese proteins are either poorly expressed in vitro, or are unstableafter expression. Recently, one copy of lig was expressed very weakly inlow passage L. krischneri RM52 (Matsunaga et al., InternationalConference on Leptospirosis, Barbados (2002). However, the expression ofLigA or LigB at the translational level in low passage strain of L.interrogans was not detected herein. The re-establishment of LigA andLigB expression upon infection and the absence of these genes innon-pathogenic leptospires suggests that they are virulence factors ofpathogenic leptospires.

The currently available whole cell leptospiral vaccines elicit ashort-term immunity. Moreover, they are ineffective in cross protectionagainst different serovars. Thus, vaccinated dogs may contractleptospirosis by the same or different serovars (Brown et al., J. Am.Vet. Med. Assoc., 209:1265-1267 (1996); Cole et al., J. Am. Vet. Med.Assoc., 180:435-437 (1982); Everard et al., Trop. Geogr. Med.,19:126-132 (1987); Harkin and Gargell, J. Am. Anim. Hosp. Assoc.,32:495-501 (1996)). Currently available serological tests are unable todiscriminate between vaccine induced leptospiral antibodies and thosedue to infection. A high MAT titer indicates infection, but a high titermay also be achieved by subsequent vaccination (Goddard et al., Vet.Microbiol., 26:191-201 (1991)). Additionally, MAT is a reliable test fordiagnosis, but it is mainly focused on major serovars such as Pomona,Grippotyphosa, Canicola, Icterohaemorragiae and Hardjo. Therefore, thereis a need for a diagnostic reagent based on antigens that are expressedonly during infection to identify animals that contract leptospirosisdespite vaccination. Moreover, the cross-reactivity within variouspathogenic serovars in the MAT needs to be validated.

Some of the MAT positive sera having a low MAT titer value of less than200 to the above-mentioned serovars of Leptospira had high KELA valuesin the KELA assay disclosed herein that uses recombinant antigens ofLigA and LigB. Further analyses of these sera for other serovars such asAutmnalis and Bratislava showed high MAT titer value. The KELA disclosedherein having recombinant antigens from the conserved region of LigA andLigB thus can be utilized as a diagnostic tool for leptospirosis.

KELA with recombinant antigens to the conserved regions of LigA and LigB(Con) showed stronger reactivity than VarA and VarB to MAT positivecanine sera (Table 4). The overall sensitivity of the recombinantantigens Con, VarA and VarB to MAT positive canine sera was 58%, 33% and17%, respectively.

In conclusion, the KELA disclosed herein using recombinant LigA and LigBantigen is specific for the serodiagnosis of leptospiral infection. Thelack of antibodies to the recombinant LigA and LigB antigens invaccinated sera suggests that these antigens can be used to identifynatural infection of leptospirosis despite vaccination.

Example V Use of LigA Protein as a Vaccine Candidate Against Infectionby L. interrogans Serovar Pomona

The cloning of LigA protein of Leptospira interrogans was previouslyreported. The potential use of this protein as a vaccine candidateagainst infection by L. interroigans serovar Pomona was evaluatedherein. LigA was truncated into the N-terminal 599 amino acids conservedregion (Con, without the signal sequence) and the C-terminal 595 aminoacid variable region of LigA (VarA) and expressed in Escherichia coli asa fusion protein with glutathione-S-transferase (rCon-GST andrVarA-GST). Eight Golden Syrian hamsters were vaccinated with rCon-GSTand rVarA-GST along with adjuvant aluminum hydroxide at 4 and 8 week ofage. Sixteen hamsters were used as nonvaccinated controls withGST-adjuvant (8 animals) and adjuvant (8 animals). Three weeks after thelast vaccination, all animals were intraperitoneally inoculated with 108of L. interrogans serovar Pomona (NVSL 1427-35-093002). All eightvaccinated hamsters survived after challenge. In contrast, 2 or 3 out of8 animals died 2 weeks after challenge in the GST-adjuvant and adjuvantcontrol group, respectively. Hamsters in the control groups had kidneylesions and were also culture positive in various tissues, but not thevaccinated ones. RCon-GST/rVar-GST elicited an antibody response to rConin the hamsters by kinetic enzyme-linked immunosorbent assay. Resultsfrom this study show that vaccination with rCon-GST/rVar-GST protectedhamsters against infection and disease after challenge with L.interrogans serovar Pomona.

All publications, patents and patent applications referred to areincorporated herein by reference. While in the foregoing specificationthis invention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

1. A method for the detection of pathogenic Leptospira infection in amammal, comprising the steps of: a) contacting isolated LigA proteinfrom Leptospira interrogans with a biological sample from a mammalsuspected of having Leptospira infection, wherein the isolated LigAprotein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:33 or consists of 50 or more contiguous amino acids of SEQ ID NO: 2, andb) detecting the presence of a complex formed between LigA andantibodies in the biological sample, thereby indicating pathogenicLeptospira infection in the mammal.
 2. The method according to claim 1,wherein the mammal is a human, dog, bovine, porcine, or horse.
 3. Themethod of claim 1, wherein said detecting comprises: contacting thesample, after said contacting LigA protein from Leptospira interroganswith a biological sample, with an indicator component which detectscomplexes of immunologically active LigA protein and antibodies from thebiological sample.
 4. The method of claim 1, wherein the LigA proteincomprises the amino acid sequence of SEQ ID NO:
 2. 5. The methodaccording to claim 1, wherein the LigA protein comprises the amino acidsequence of SEQ ID NO:
 33. 6. The method according to claim 1, whereinthe LigA protein consists of 50 or more contiguous amino acids of SEQ IDNO:2.
 7. The method according to claim 6, wherein the LigA protein has alength of 50 to 1000 amino acids.
 8. The method as claimed in claim 3,wherein the indicator component is an antibody which is directed againstthe antibody to be detected and which has a label.
 9. The method asclaimed in claim 8, wherein the label comprises a radioactive isotope.10. The method as claimed in claim 8, wherein the label comprises anenzyme which is able to catalyze a color reaction.
 11. The method asclaimed in claim 8, wherein the immunologically active protein isbiotinylated, and the indicator component is avidin or streptavidinhaving an enzyme covalently bonded thereto.
 12. The method as claimed inclaim 8, wherein the method is carried out with an ELISA assay kit. 13.The method as claimed in claim 12, wherein the LigA protein is coupledto microtiter plates, and the indicator component comprises anti-humanimmunoglobulin to which an enzyme catalyzing a color reaction iscoupled.
 14. The method of claim 13, wherein the enzyme is a peroxidase.15. The method of claim 13, wherein the indicator component comprisesIgG antibodies, IgM antibodies or a mixture thereof.